A Versatile Microarray Platform for Capturing Rare Cells

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Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

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Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

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Boyang Cao

Full Text Available Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

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Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

2014-01-01

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp. PMID:25469776

Improvement in the amine glass platform by bubbling method for a DNA microarray.

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Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

2015-01-01

A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool.

Improvement in the amine glass platform by bubbling method for a DNA microarray

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Jee SH

2015-10-01

Full Text Available Seung Hyun Jee,1 Jong Won Kim,2 Ji Hyeong Lee,2 Young Soo Yoon11Department of Chemical and Biological Engineering, Gachon University, Seongnam, Gyeonggi, Republic of Korea; 2Genomics Clinical Research Institute, LabGenomics Co., Ltd., Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of KoreaAbstract: A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool. Keywords: DNA microarray, glass platform, bubbling method, self-assambled monolayer

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray

DEFF Research Database (Denmark)

Stolc, Viktor; Li, Lei; Wang, Xiangfeng

2005-01-01

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map...... transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference...... gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models...

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

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Palta Priit

2010-04-01

Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

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Chen Feng

2010-10-01

Full Text Available Abstract Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals and Plasmodium falciparum (a related parasite responsible for severe human malaria, we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

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Wang, Zheng; Vora, Gary J; Stenger, David A

2004-07-01

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB.

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Chatziioannou, Aristotelis; Moulos, Panagiotis; Kolisis, Fragiskos N

2009-10-27

The microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods. We describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime. Gene ARMADA provides a

Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

Energy Technology Data Exchange (ETDEWEB)

Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

2008-07-15

Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

Can subtle changes in gene expression be consistently detected with different microarray platforms?

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Kuiper Rowan

2008-03-01

Full Text Available Abstract Background The comparability of gene expression data generated with different microarray platforms is still a matter of concern. Here we address the performance and the overlap in the detection of differentially expressed genes for five different microarray platforms in a challenging biological context where differences in gene expression are few and subtle. Results Gene expression profiles in the hippocampus of five wild-type and five transgenic δC-doublecortin-like kinase mice were evaluated with five microarray platforms: Applied Biosystems, Affymetrix, Agilent, Illumina, LGTC home-spotted arrays. Using a fixed false discovery rate of 10% we detected surprising differences between the number of differentially expressed genes per platform. Four genes were selected by ABI, 130 by Affymetrix, 3,051 by Agilent, 54 by Illumina, and 13 by LGTC. Two genes were found significantly differentially expressed by all platforms and the four genes identified by the ABI platform were found by at least three other platforms. Quantitative RT-PCR analysis confirmed 20 out of 28 of the genes detected by two or more platforms and 8 out of 15 of the genes detected by Agilent only. We observed improved correlations between platforms when ranking the genes based on the significance level than with a fixed statistical cut-off. We demonstrate significant overlap in the affected gene sets identified by the different platforms, although biological processes were represented by only partially overlapping sets of genes. Aberrances in GABA-ergic signalling in the transgenic mice were consistently found by all platforms. Conclusion The different microarray platforms give partially complementary views on biological processes affected. Our data indicate that when analyzing samples with only subtle differences in gene expression the use of two different platforms might be more attractive than increasing the number of replicates. Commercial two-color platforms seem to

Comparison of Comparative Genomic Hybridization Technologies across Microarray Platforms

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In the 2007 Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation (CNV) was analyzed ...

Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.

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Mannion James W

2002-10-01

Full Text Available Abstract Background Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG 3 days following sciatic nerve transection (axotomy. Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy. Results Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P 1.5-fold expression change and P 1.5-fold and P in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567. Temporal patterns of individual genes regulation varied. Conclusions We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.

Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

Science.gov (United States)

Golova, Julia B.; Chernov, Boris K.

2010-04-27

New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

A dynamic bead-based microarray for parallel DNA detection

International Nuclear Information System (INIS)

Sochol, R D; Lin, L; Casavant, B P; Dueck, M E; Lee, L P

2011-01-01

A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm 2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

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Sascha D Braun

Full Text Available Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL and narrow spectrum (NSBL beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13, blaGIM (2/2, blaKPC (27/27, blaNDM (5/5, blaIMP-2/4/7/8/13/14/15/16/31 (10/10, blaOXA-23 (12/13, blaOXA-40-group (7/7, blaOXA-48-group (32/33, blaOXA-51 (1/1 and blaOXA-58 (1/1. Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16, blaOXA-2 (4/4, blaOXA-9 (33/33, OXA-10 (3/3, blaOXA-51 (25/25, blaOXA-58 (2/2, CTX-M1/M15 (17/17 and blaVIM (1/1]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4% isolates, including Acinetobacter baumannii (28/28, Enterobacter spec. (5/5, Escherichia coli (4/4, Klebsiella pneumoniae (62/63, Klebsiella oxytoca (0/2, Pseudomonas aeruginosa (12/12, Citrobacter freundii (1/1 and Citrobacter

High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

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Magness Charles L

2007-01-01

Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

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Sebastian Boltaña

2017-02-01

Full Text Available This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS and peptidoglycan (PGN. Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs, carrier proteins/membrane transport (approximately 15%, effectors/modulators and cell communication (approximately 11%, nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5% and intracellular transducers/signal transduction (approximately 5%. Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ that provides a platform enriched for the study

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

Science.gov (United States)

Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

2017-02-03

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene

Position dependent mismatch discrimination on DNA microarrays – experiments and model

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Michel Wolfgang

2008-12-01

Full Text Available Abstract Background The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study. Results We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results. Conclusion Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.

Microarray-based ultra-high resolution discovery of genomic deletion mutations

Science.gov (United States)

2014-01-01

Background Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. Results Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. Conclusions Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence. PMID:24655320

Plant-pathogen interactions: what microarray tells about it?

Science.gov (United States)

Lodha, T D; Basak, J

2012-01-01

Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

Science.gov (United States)

Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that

LNA-modified isothermal oligonucleotide microarray for ...

Indian Academy of Sciences (India)

2014-10-20

Oct 20, 2014 ... the advent of DNA microarray techniques (Lee et al. 2007). ... atoms of ribose to form a bicyclic ribosyl structure. It is the .... 532 nm and emission at 570 nm. The signal ..... sis and validation using real-time PCR. Nucleic Acids ...

Preparation of oligonucleotide microarray for radiation-associated gene expression detection and its application in lung cancer cell lines

International Nuclear Information System (INIS)

Guo Wanfeng; Lin Ruxian; Huang Jian; Guo Guozhen; Wang Shengqi

2005-01-01

Objective: The response of tumor cell to radiation is accompanied by complex change in patterns of gene expression. It is highly probable that a better understanding of molecular and genetic changes can help to sensitize the radioresistant tumor cells. Methods: Oligonucleotide microarray provides a powerful tool for high-throughput identifying a wider range of genes involved in the radioresistance. Therefore, the authors designed one oligonucleotide microarray according to the biological effect of IR. By using different radiosensitive lung cancer cell lines, the authors identified genes showing altered expression in lung cancer cell lines. To provide independent confirmation of microarray data, semi-quantitative RT-PCR was performed on a selection of genes. Results: In radioresistant A549 cell lines, a total of 18 genes were selected as having significant fold-changes compared to NCI-H446, 8 genes were up-regulated and 10 genes were down-regulated. Subsequently, A549 and NCI-H446 cells were delivered by ionizing radiation. In A549 cell line, we found 22 (19 up-regulated and 3 down-regulated) and 26 (8 up-regulated and 18 down-regulated) differentially expressed genes at 6h and 24h after ionizing radiation. In NCI-H446 cell line, we identified 17 (9 up-regulated and 8 down-regulated) and 18 (6 up-regulated and 12 down-regulated) differentially expressed genes at 6 h and 24 h after ionizing radiation. The authors tested seven genes (MDM2, p53, XRCC5, Bcl-2, PIM2, NFKBIA and Cyclin B1) for RT-PCR, and found that the results were in good agreement with those from the microarray data except for NFKBIA gene, even though the value for each mRNA level might be different between the two measurements. In present study, the authors identified some genes with cell proliferation and anti-apoptosis, such as MdM2, BCL-2, PKCz and PIM2 expression levels increased in A549 cells and decreased in NCI-H446 cells after radiation, and other genes with DNA repair, such as XRCC5, ERCC5

Comparative analysis of methods for gene transcription profiling data derived from different microarray technologies in rat and mouse models of diabetes

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Bihoreau Marie-Thérèse

2009-02-01

Full Text Available Abstract Background Microarray technologies are widely used to quantify the abundance of transcripts corresponding to thousands of genes. To maximise the robustness of transcriptome results, we have tested the performance and reproducibility of rat and mouse gene expression data obtained with Affymetrix, Illumina and Operon platforms. Results We present a thorough analysis of the degree of reproducibility provided by analysing the transcriptomic profile of the same animals of several experimental groups under different popular microarray technologies in different tissues. Concordant results from inter- and intra-platform comparisons were maximised by testing many popular computational methods for generating fold changes and significances and by only considering oligonucleotides giving high expression levels. The choice of Affymetrix signal extraction technique was shown to have the greatest effect on the concordance across platforms. In both species, when choosing optimal methods, the agreement between data generated on the Affymetrix and Illumina was excellent; this was verified using qRT-PCR on a selection of genes present on all platforms. Conclusion This study provides an extensive assessment of analytical methods best suited for processing data from different microarray technologies and can assist integration of technologically different gene expression datasets in biological systems.

Combining gene expression data from different generations of oligonucleotide arrays

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Kong Sek

2004-10-01

Full Text Available Abstract Background One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. Results We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. Conclusions Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different

Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

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Dial Stacey L

2008-07-01

Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates

KAUST Repository

Boopathi, Pon Arunachalam

2016-10-09

High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60 mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n =14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n = 85) present in the arrays showed perfect correlation (r(2) = 0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r >= 0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates. (C) 2016 Published by Elsevier B.V.

Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates

KAUST Repository

Boopathi, Pon Arunachalam; Subudhi, Amit; Middha, Sheetal; Acharya, Jyoti; Mugasimangalam, Raja Chinnadurai; Kochar, Sanjay Kumar; Kochar, Dhanpat Kumar; Das, Ashis

2016-01-01

High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60 mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n =14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n = 85) present in the arrays showed perfect correlation (r(2) = 0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r >= 0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates. (C) 2016 Published by Elsevier B.V.

Comparison of microarray platforms for measuring differential microRNA expression in paired normal/cancer colon tissues.

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Maurizio Callari

Full Text Available BACKGROUND: Microarray technology applied to microRNA (miRNA profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi, comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport. CONCLUSIONS: Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs - gene expression integration approach.

The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays

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Brazma Alvis

2010-03-01

Full Text Available Abstract Background Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results The IronChip Evaluation Package (ICEP is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see "Additional Files" section and at: http://www.alice-dsl.net/evgeniy.vainshtein/ICEP/

Cross-platform comparison of microarray data using order restricted inference

Science.gov (United States)

Klinglmueller, Florian; Tuechler, Thomas; Posch, Martin

2013-01-01

Motivation Titration experiments measuring the gene expression from two different tissues, along with total RNA mixtures of the pure samples, are frequently used for quality evaluation of microarray technologies. Such a design implies that the true mRNA expression of each gene, is either constant or follows a monotonic trend between the mixtures, applying itself to the use of order restricted inference procedures. Exploiting only the postulated monotonicity of titration designs, we propose three statistical analysis methods for the validation of high-throughput genetic data and corresponding preprocessing techniques. Results Our methods allow for inference of accuracy, repeatability and cross-platform agreement, with minimal required assumptions regarding the underlying data generating process. Therefore, they are readily applicable to all sorts of genetic high-throughput data independent of the degree of preprocessing. An application to the EMERALD dataset was used to demonstrate how our methods provide a rich spectrum of easily interpretable quality metrics and allow the comparison of different microarray technologies and normalization methods. The results are on par with previous work, but provide additional new insights that cast doubt on the utility of popular preprocessing techniques, specifically concerning the EMERALD projects dataset. Availability All datasets are available on EBI’s ArrayExpress web site (http://www.ebi.ac.uk/microarray-as/ae/) under accession numbers E-TABM-536, E-TABM-554 and E-TABM-555. Source code implemented in C and R is available at: http://statistics.msi.meduniwien.ac.at/float/cross_platform/. Methods for testing and variance decomposition have been made available in the R-package orQA, which can be downloaded and installed from CRAN http://cran.r-project.org. PMID:21317143

Microarray labeling extension values: laboratory signatures for Affymetrix GeneChips

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Lee, Yun-Shien; Chen, Chun-Houh; Tsai, Chi-Neu; Tsai, Chia-Lung; Chao, Angel; Wang, Tzu-Hao

2009-01-01

Interlaboratory comparison of microarray data, even when using the same platform, imposes several challenges to scientists. RNA quality, RNA labeling efficiency, hybridization procedures and data-mining tools can all contribute variations in each laboratory. In Affymetrix GeneChips, about 11–20 different 25-mer oligonucleotides are used to measure the level of each transcript. Here, we report that ‘labeling extension values (LEVs)’, which are correlation coefficients between probe intensities and probe positions, are highly correlated with the gene expression levels (GEVs) on eukayotic Affymetrix microarray data. By analyzing LEVs and GEVs in the publicly available 2414 cel files of 20 Affymetrix microarray types covering 13 species, we found that correlations between LEVs and GEVs only exist in eukaryotic RNAs, but not in prokaryotic ones. Surprisingly, Affymetrix results of the same specimens that were analyzed in different laboratories could be clearly differentiated only by LEVs, leading to the identification of ‘laboratory signatures’. In the examined dataset, GSE10797, filtering out high-LEV genes did not compromise the discovery of biological processes that are constructed by differentially expressed genes. In conclusion, LEVs provide a new filtering parameter for microarray analysis of gene expression and it may improve the inter- and intralaboratory comparability of Affymetrix GeneChips data. PMID:19295132

Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification.

Science.gov (United States)

Oberthuer, André; Berthold, Frank; Warnat, Patrick; Hero, Barbara; Kahlert, Yvonne; Spitz, Rüdiger; Ernestus, Karen; König, Rainer; Haas, Stefan; Eils, Roland; Schwab, Manfred; Brors, Benedikt; Westermann, Frank; Fischer, Matthias

2006-11-01

To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

Science.gov (United States)

2013-01-01

Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

Science.gov (United States)

Bae, Jin-Woo; Park, Yong-Ha

2006-07-01

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

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Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

2014-01-01

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

Changes in the peripheral blood transcriptome associated with occupational benzene exposure identified by cross-comparison on two microarray platforms

Energy Technology Data Exchange (ETDEWEB)

McHale, Cliona M.; Zhang, Luoping; Lan, Qing; Li, Guilan; Hubbard, Alan E.; Forrest, Matthew S.; Vermeulen, Roel; Chen, Jinsong; Shen, Min; Rappaport, Stephen M.; Yin, Songnian; Smith, Martyn T.; Rothman, Nathaniel

2009-03-01

Benzene is an established cause of leukemia and a possible cause of lymphoma in humans but the molecular pathways underlying this remain largely undetermined. This study sought to determine if the use of two different microarray platforms could identify robust global gene expression and pathway changes associated with occupational benzene exposure in the peripheral blood mononuclear cell (PBMC) gene expression of a population of shoe-factory workers with well-characterized occupational exposures to benzene. Microarray data was analyzed by a robust t-test using a Quantile Transformation (QT) approach. Differential expression of 2692 genes using the Affymetrix platform and 1828 genes using the Illumina platform was found. While the overall concordance in genes identified as significantly associated with benzene exposure between the two platforms was 26% (475 genes), the most significant genes identified by either array were more likely to be ranked as significant by the other platform (Illumina = 64%, Affymetrix = 58%). Expression ratios were similar among the concordant genes (mean difference in expression ratio = 0.04, standard deviation = 0.17). Four genes (CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were identified by both platforms in the current study and were among the top 100 genes. Gene Ontology analysis showed over representation of genes involved in apoptosis among the concordant genes while Ingenuity{reg_sign} Pathway Analysis (IPA) identified pathways related to lipid metabolism. Using a two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.

A High-Throughput Antibody-Based Microarray Typing Platform

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Ashan Perera

2013-05-01

Full Text Available Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing E. coli (STEC as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers, this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

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Niggli Felix K

2006-06-01

Full Text Available Abstract Background The Epstein-Barr virus (EBV is associated with lymphoid malignancies, including Burkitt's lymphoma (BL, and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. Results To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2, and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2, and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. Conclusion Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology.

PCR amplification on microarrays of gel immobilized oligonucleotides

Science.gov (United States)

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

Direct calibration of PICKY-designed microarrays

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Ronald Pamela C

2009-10-01

Full Text Available Abstract Background Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples. Results Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu under the PICKY Download area. Conclusion PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.

Detection of genomic deletions in rice using oligonucleotide microarrays

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Bordeos Alicia

2009-03-01

Full Text Available Abstract Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL. However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/. Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a

Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

DEFF Research Database (Denmark)

Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

2015-01-01

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...

PIIKA 2: an expanded, web-based platform for analysis of kinome microarray data.

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Brett Trost

Full Text Available Kinome microarrays are comprised of peptides that act as phosphorylation targets for protein kinases. This platform is growing in popularity due to its ability to measure phosphorylation-mediated cellular signaling in a high-throughput manner. While software for analyzing data from DNA microarrays has also been used for kinome arrays, differences between the two technologies and associated biologies previously led us to develop Platform for Intelligent, Integrated Kinome Analysis (PIIKA, a software tool customized for the analysis of data from kinome arrays. Here, we report the development of PIIKA 2, a significantly improved version with new features and improvements in the areas of clustering, statistical analysis, and data visualization. Among other additions to the original PIIKA, PIIKA 2 now allows the user to: evaluate statistically how well groups of samples cluster together; identify sets of peptides that have consistent phosphorylation patterns among groups of samples; perform hierarchical clustering analysis with bootstrapping; view false negative probabilities and positive and negative predictive values for t-tests between pairs of samples; easily assess experimental reproducibility; and visualize the data using volcano plots, scatterplots, and interactive three-dimensional principal component analyses. Also new in PIIKA 2 is a web-based interface, which allows users unfamiliar with command-line tools to easily provide input and download the results. Collectively, the additions and improvements described here enhance both the breadth and depth of analyses available, simplify the user interface, and make the software an even more valuable tool for the analysis of kinome microarray data. Both the web-based and stand-alone versions of PIIKA 2 can be accessed via http://saphire.usask.ca.

PIIKA 2: an expanded, web-based platform for analysis of kinome microarray data.

Science.gov (United States)

Trost, Brett; Kindrachuk, Jason; Määttänen, Pekka; Napper, Scott; Kusalik, Anthony

2013-01-01

Kinome microarrays are comprised of peptides that act as phosphorylation targets for protein kinases. This platform is growing in popularity due to its ability to measure phosphorylation-mediated cellular signaling in a high-throughput manner. While software for analyzing data from DNA microarrays has also been used for kinome arrays, differences between the two technologies and associated biologies previously led us to develop Platform for Intelligent, Integrated Kinome Analysis (PIIKA), a software tool customized for the analysis of data from kinome arrays. Here, we report the development of PIIKA 2, a significantly improved version with new features and improvements in the areas of clustering, statistical analysis, and data visualization. Among other additions to the original PIIKA, PIIKA 2 now allows the user to: evaluate statistically how well groups of samples cluster together; identify sets of peptides that have consistent phosphorylation patterns among groups of samples; perform hierarchical clustering analysis with bootstrapping; view false negative probabilities and positive and negative predictive values for t-tests between pairs of samples; easily assess experimental reproducibility; and visualize the data using volcano plots, scatterplots, and interactive three-dimensional principal component analyses. Also new in PIIKA 2 is a web-based interface, which allows users unfamiliar with command-line tools to easily provide input and download the results. Collectively, the additions and improvements described here enhance both the breadth and depth of analyses available, simplify the user interface, and make the software an even more valuable tool for the analysis of kinome microarray data. Both the web-based and stand-alone versions of PIIKA 2 can be accessed via http://saphire.usask.ca.

Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs).

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Turkec, Aydin; Lucas, Stuart J; Karacanli, Burçin; Baykut, Aykut; Yuksel, Hakki

2016-03-01

Detection of GMO material in crop and food samples is the primary step in GMO monitoring and regulation, with the increasing number of GM events in the world market requiring detection solutions with high multiplexing capacity. In this study, we test the suitability of a high-density oligonucleotide microarray platform for direct, quantitative detection of GMOs found in the Turkish feed market. We tested 1830 different 60nt probes designed to cover the GM cassettes from 12 different GM cultivars (3 soya, 9 maize), as well as plant species-specific and contamination controls, and developed a data analysis method aiming to provide maximum throughput and sensitivity. The system was able specifically to identify each cultivar, and in 10/12 cases was sensitive enough to detect GMO DNA at concentrations of ⩽1%. These GMOs could also be quantified using the microarray, as their fluorescence signals increased linearly with GMO concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prognostic meta-signature of breast cancer developed by two-stage mixture modeling of microarray data

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Ghosh Debashis

2004-12-01

Full Text Available Abstract Background An increasing number of studies have profiled tumor specimens using distinct microarray platforms and analysis techniques. With the accumulating amount of microarray data, one of the most intriguing yet challenging tasks is to develop robust statistical models to integrate the findings. Results By applying a two-stage Bayesian mixture modeling strategy, we were able to assimilate and analyze four independent microarray studies to derive an inter-study validated "meta-signature" associated with breast cancer prognosis. Combining multiple studies (n = 305 samples on a common probability scale, we developed a 90-gene meta-signature, which strongly associated with survival in breast cancer patients. Given the set of independent studies using different microarray platforms which included spotted cDNAs, Affymetrix GeneChip, and inkjet oligonucleotides, the individually identified classifiers yielded gene sets predictive of survival in each study cohort. The study-specific gene signatures, however, had minimal overlap with each other, and performed poorly in pairwise cross-validation. The meta-signature, on the other hand, accommodated such heterogeneity and achieved comparable or better prognostic performance when compared with the individual signatures. Further by comparing to a global standardization method, the mixture model based data transformation demonstrated superior properties for data integration and provided solid basis for building classifiers at the second stage. Functional annotation revealed that genes involved in cell cycle and signal transduction activities were over-represented in the meta-signature. Conclusion The mixture modeling approach unifies disparate gene expression data on a common probability scale allowing for robust, inter-study validated prognostic signatures to be obtained. With the emerging utility of microarrays for cancer prognosis, it will be important to establish paradigms to meta

Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors

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Naume B

2008-08-01

Full Text Available Abstract Background Microarray Comparative Genomic Hybridization (array CGH provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes. Results We evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from http://www.ifi.uio.no/bioinf/Projects/. Conclusion We conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.

A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen.

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Lee, SangWook; Lee, Jong Hyun; Kwon, Hyuck Gi; Laurell, Thomas; Jeong, Ok Chan; Kim, Soyoun

2018-01-01

Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate?specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol?gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.

A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants

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Pląder Wojciech

2011-09-01

Full Text Available Abstract Plastids are small organelles equipped with their own genomes (plastomes. Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile.

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

International Nuclear Information System (INIS)

Estilo, Cherry L; Boyle, Jay O; Kraus, Dennis H; Patel, Snehal; Shaha, Ashok R; Wong, Richard J; Huryn, Joseph M; Shah, Jatin P; Singh, Bhuvanesh; O-charoenrat, Pornchai; Talbot, Simon; Socci, Nicholas D; Carlson, Diane L; Ghossein, Ronald; Williams, Tijaana; Yonekawa, Yoshihiro; Ramanathan, Yegnanarayana

2009-01-01

The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling. The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG-U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR. Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by

DNA microarray technique for detecting food-borne pathogens

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Xing GAO

2012-08-01

Full Text Available Objective To study the application of DNA microarray technique for screening and identifying multiple food-borne pathogens. Methods The oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogens, and then were validated by bioinformatic analyses. The 5' end of each probe was modified by amino-group and 10 Poly-T, and the optimized probes were synthesized and spotted on aldehyde-coated slides. The bacteria DNA template incubated with Klenow enzyme was amplified by arbitrarily primed PCR, and PCR products incorporated into Aminoallyl-dUTP were coupled with fluorescent dye. After hybridization of the purified PCR products with DNA microarray, the hybridization image and fluorescence intensity analysis was acquired by ScanArray and GenePix Pro 5.1 software. A series of detection conditions such as arbitrarily primed PCR and microarray hybridization were optimized. The specificity of this approach was evaluated by 16 different bacteria DNA, and the sensitivity and reproducibility were verified by 4 food-borne pathogens DNA. The samples of multiple bacteria DNA and simulated water samples of Shigella dysenteriae were detected. Results Nine different food-borne bacteria were successfully discriminated under the same condition. The sensitivity of genomic DNA was 102 －103pg/ μl, and the coefficient of variation (CV of the reproducibility of assay was less than 15%. The corresponding specific hybridization maps of the multiple bacteria DNA samples were obtained, and the detection limit of simulated water sample of Shigella dysenteriae was 3.54×105cfu/ml. Conclusions The DNA microarray detection system based on arbitrarily primed PCR can be employed for effective detection of multiple food-borne pathogens, and this assay may offer a new method for high-throughput platform for detecting bacteria.

Uses of Dendrimers for DNA Microarrays

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Jean-Pierre Majoral

2006-08-01

Full Text Available Biosensors such as DNA microarrays and microchips are gaining an increasingimportance in medicinal, forensic, and environmental analyses. Such devices are based onthe detection of supramolecular interactions called hybridizations that occur betweencomplementary oligonucleotides, one linked to a solid surface (the probe, and the other oneto be analyzed (the target. This paper focuses on the improvements that hyperbranched andperfectly defined nanomolecules called dendrimers can provide to this methodology. Twomain uses of dendrimers for such purpose have been described up to now; either thedendrimer is used as linker between the solid surface and the probe oligonucleotide, or thedendrimer is used as a multilabeled entity linked to the target oligonucleotide. In the firstcase the dendrimer generally induces a higher loading of probes and an easier hybridization,due to moving away the solid phase. In the second case the high number of localized labels(generally fluorescent induces an increased sensitivity, allowing the detection of smallquantities of biological entities.

A flexible whole-genome microarray for transcriptomics in three-spine stickleback (Gasterosteus aculeatus

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Primmer Craig R

2009-09-01

Full Text Available Abstract Background The use of microarray technology for describing changes in mRNA expression to address ecological and evolutionary questions is becoming increasingly popular. Since three-spine stickleback are an important ecological and evolutionary model-species as well as an emerging model for eco-toxicology, the ability to have a functional and flexible microarray platform for transcriptome studies will greatly enhance the research potential in these areas. Results We designed 43,392 unique oligonucleotide probes representing 19,274 genes (93% of the estimated total gene number, and tested the hybridization performance of both DNA and RNA from different populations to determine the efficacy of probe design for transcriptome analysis using the Agilent array platform. The majority of probes were functional as evidenced by the DNA hybridization success, and 30,946 probes (14,615 genes had a signal that was significantly above background for RNA isolated from liver tissue. Genes identified as being expressed in liver tissue were grouped into functional categories for each of the three Gene Ontology groups: biological process, molecular function, and cellular component. As expected, the highest proportions of functional categories belonged to those associated with metabolic functions: metabolic process, binding, catabolism, and organelles. Conclusion The probe and microarray design presented here provides an important step facilitating transcriptomics research for this important research organism by providing a set of over 43,000 probes whose hybridization success and specificity to liver expression has been demonstrated. Probes can easily be added or removed from the current design to tailor the array to specific experiments and additional flexibility lies in the ability to perform either one-color or two-color hybridizations.

Performance comparison of two microarray platforms to assess differential gene expression in human monocyte and macrophage cells

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Montalescot Gilles

2008-06-01

Full Text Available Abstract Background In this study we assessed the respective ability of Affymetrix and Illumina microarray methodologies to answer a relevant biological question, namely the change in gene expression between resting monocytes and macrophages derived from these monocytes. Five RNA samples for each type of cell were hybridized to the two platforms in parallel. In addition, a reference list of differentially expressed genes (DEG was generated from a larger number of hybridizations (mRNA from 86 individuals using the RNG/MRC two-color platform. Results Our results show an important overlap of the Illumina and Affymetrix DEG lists. In addition, more than 70% of the genes in these lists were also present in the reference list. Overall the two platforms had very similar performance in terms of biological significance, evaluated by the presence in the DEG lists of an excess of genes belonging to Gene Ontology (GO categories relevant for the biology of monocytes and macrophages. Our results support the conclusion of the MicroArray Quality Control (MAQC project that the criteria used to constitute the DEG lists strongly influence the degree of concordance among platforms. However the importance of prioritizing genes by magnitude of effect (fold change rather than statistical significance (p-value to enhance cross-platform reproducibility recommended by the MAQC authors was not supported by our data. Conclusion Functional analysis based on GO enrichment demonstrates that the 2 compared technologies delivered very similar results and identified most of the relevant GO categories enriched in the reference list.

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

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Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario

2007-05-01

To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that

The Plasmodium falciparum Sexual Development Transcriptome: A Microarray Analysis using Ontology-Based Pattern Identification

National Research Council Canada - National Science Library

Young, Jason A; Fivelman, Quinton L; Blair, Peter L; de la Vega, Patricia; Le Roch, Karine G; Zhou, Yingyao; Carucci, Daniel J; Baker, David A; Winzeler, Elizabeth A

2005-01-01

... a full-genome high-density oligonucleotide microarray. The interpretation of this transcriptional data was aided by applying a novel knowledge-based data-mining algorithm termed ontology-based pattern identification (OPI...

The use of microarrays in microbial ecology

Energy Technology Data Exchange (ETDEWEB)

Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

2009-09-15

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

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Bidard, Frédérique; Imbeaud, Sandrine; Reymond, Nancie; Lespinet, Olivier; Silar, Philippe; Clavé, Corinne; Delacroix, Hervé; Berteaux-Lecellier, Véronique; Debuchy, Robert

2010-06-18

The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

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Bidard Frédérique

2010-06-01

Full Text Available Abstract Background The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. Findings We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS, we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. Conclusions A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

Facilitating RNA structure prediction with microarrays.

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Kierzek, Elzbieta; Kierzek, Ryszard; Turner, Douglas H; Catrina, Irina E

2006-01-17

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia.

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Schönmann, Susan; Loy, Alexander; Wimmersberger, Céline; Sobek, Jens; Aquino, Catharine; Vandamme, Peter; Frey, Beat; Rehrauer, Hubert; Eberl, Leo

2009-04-01

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

Science.gov (United States)

Samolski, Ilanit; de Luis, Alberto; Vizcaíno, Juan Antonio; Monte, Enrique; Suárez, M Belén

2009-10-13

It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as

Direct microcontact printing of oligonucleotides for biochip applications

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Trévisiol E

2005-07-01

Full Text Available Abstract Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting

Improving the scaling normalization for high-density oligonucleotide GeneChip expression microarrays

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Lu Chao

2004-07-01

Full Text Available Abstract Background Normalization is an important step for microarray data analysis to minimize biological and technical variations. Choosing a suitable approach can be critical. The default method in GeneChip expression microarray uses a constant factor, the scaling factor (SF, for every gene on an array. The SF is obtained from a trimmed average signal of the array after excluding the 2% of the probe sets with the highest and the lowest values. Results Among the 76 U34A GeneChip experiments, the total signals on each array showed 25.8% variations in terms of the coefficient of variation, although all microarrays were hybridized with the same amount of biotin-labeled cRNA. The 2% of the probe sets with the highest signals that were normally excluded from SF calculation accounted for 34% to 54% of the total signals (40.7% ± 4.4%, mean ± sd. In comparison with normalization factors obtained from the median signal or from the mean of the log transformed signal, SF showed the greatest variation. The normalization factors obtained from log transformed signals showed least variation. Conclusions Eliminating 40% of the signal data during SF calculation failed to show any benefit. Normalization factors obtained with log transformed signals performed the best. Thus, it is suggested to use the mean of the logarithm transformed data for normalization, rather than the arithmetic mean of signals in GeneChip gene expression microarrays.

Correction of Spatial Bias in Oligonucleotide Array Data

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Philippe Serhal

2013-01-01

Full Text Available Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs for each intended target, on average, correlate with their target’s true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users’ current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays. A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias.

Correction of Spatial Bias in Oligonucleotide Array Data

Science.gov (United States)

Lemieux, Sébastien

2013-01-01

Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs) for each intended target, on average, correlate with their target's true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users' current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays). A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias. PMID:23573083

Simulation of microarray data with realistic characteristics

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Lehmussola Antti

2006-07-01

Full Text Available Abstract Background Microarray technologies have become common tools in biological research. As a result, a need for effective computational methods for data analysis has emerged. Numerous different algorithms have been proposed for analyzing the data. However, an objective evaluation of the proposed algorithms is not possible due to the lack of biological ground truth information. To overcome this fundamental problem, the use of simulated microarray data for algorithm validation has been proposed. Results We present a microarray simulation model which can be used to validate different kinds of data analysis algorithms. The proposed model is unique in the sense that it includes all the steps that affect the quality of real microarray data. These steps include the simulation of biological ground truth data, applying biological and measurement technology specific error models, and finally simulating the microarray slide manufacturing and hybridization. After all these steps are taken into account, the simulated data has realistic biological and statistical characteristics. The applicability of the proposed model is demonstrated by several examples. Conclusion The proposed microarray simulation model is modular and can be used in different kinds of applications. It includes several error models that have been proposed earlier and it can be used with different types of input data. The model can be used to simulate both spotted two-channel and oligonucleotide based single-channel microarrays. All this makes the model a valuable tool for example in validation of data analysis algorithms.

Comparison of gene expression of mitogenic kinin path in adherent and non-adherent CD 34-stem cells using oligonucleotide microarrays.

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Krzysztof Machaj

2008-02-01

Full Text Available One of the more interesting cells present in the umbilical cord blood - as far as their potential clinical use is concerned - are stem cells not presenting the CD34 antigen. These are the pluripotential cells with their biological properties similar to mesenchymal stem cells, with the ability to differentiate into such tissue types as bone, cartilage, nervous (to some extent, glia and muscle. The authors compared the activity of genes coding the proteins in mitogenic signal paths activated by kinin receptors using oligonucleotide microarrays in adherent and non-adherent CD 34- cells derived from umbilical cord blood. In the linear regression model with a 95% prognosis area for differentiating genes outside this area, the following genes were selected: c-jun (present in 3 isoforms and c-fos. The fos and jun genes create the AP-1 transcriptive factor which regulates the expression of genes taking part in numerous cellular processes, including the cell cycle and mitosis. The obtained results shed some light on the molecular processes behind the MSC proliferation and are a starting point for further studies on the mesenchymal stem cell biology.

Gene Expression Profiling and Identification of Resistance Genes to Aspergillus flavus Infection in Peanut through EST and Microarray Strategies

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Baozhu Guo

2011-06-01

Full Text Available Aspergillus flavus and A. parasiticus infect peanut seeds and produce aflatoxins, which are associated with various diseases in domestic animals and humans throughout the world. The most cost-effective strategy to minimize aflatoxin contamination involves the development of peanut cultivars that are resistant to fungal infection and/or aflatoxin production. To identify peanut Aspergillus-interactive and peanut Aspergillus-resistance genes, we carried out a large scale peanut Expressed Sequence Tag (EST project which we used to construct a peanut glass slide oligonucleotide microarray. The fabricated microarray represents over 40% of the protein coding genes in the peanut genome. For expression profiling, resistant and susceptible peanut cultivars were infected with a mixture of Aspergillus flavus and parasiticus spores. The subsequent microarray analysis identified 62 genes in resistant cultivars that were up-expressed in response to Aspergillus infection. In addition, we identified 22 putative Aspergillus-resistance genes that were constitutively up-expressed in the resistant cultivar in comparison to the susceptible cultivar. Some of these genes were homologous to peanut, corn, and soybean genes that were previously shown to confer resistance to fungal infection. This study is a first step towards a comprehensive genome-scale platform for developing Aspergillus-resistant peanut cultivars through targeted marker-assisted breeding and genetic engineering.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

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The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA

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Chan Alan

2006-06-01

Full Text Available Abstract Background Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. Results In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A. Conclusion Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA.

Science.gov (United States)

Gibbons, Brian; Datta, Parikkhit; Wu, Ying; Chan, Alan; Al Armour, John

2006-06-30

Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH) we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A). Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

Microarray analysis identified Puccinia striiformis f. sp. tritici genes involved in infection and sporulation.

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Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, one of the most important diseases of wheat worldwide. To identify Pst genes involved in infection and sporulation, a custom oligonucleotide Genechip was made using sequences of 442 genes selected from Pst cDNA libraries. Microarray analy...

Assessing Bacterial Interactions Using Carbohydrate-Based Microarrays

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Andrea Flannery

2015-12-01

Full Text Available Carbohydrates play a crucial role in host-microorganism interactions and many host glycoconjugates are receptors or co-receptors for microbial binding. Host glycosylation varies with species and location in the body, and this contributes to species specificity and tropism of commensal and pathogenic bacteria. Additionally, bacterial glycosylation is often the first bacterial molecular species encountered and responded to by the host system. Accordingly, characterising and identifying the exact structures involved in these critical interactions is an important priority in deciphering microbial pathogenesis. Carbohydrate-based microarray platforms have been an underused tool for screening bacterial interactions with specific carbohydrate structures, but they are growing in popularity in recent years. In this review, we discuss carbohydrate-based microarrays that have been profiled with whole bacteria, recombinantly expressed adhesins or serum antibodies. Three main types of carbohydrate-based microarray platform are considered; (i conventional carbohydrate or glycan microarrays; (ii whole mucin microarrays; and (iii microarrays constructed from bacterial polysaccharides or their components. Determining the nature of the interactions between bacteria and host can help clarify the molecular mechanisms of carbohydrate-mediated interactions in microbial pathogenesis, infectious disease and host immune response and may lead to new strategies to boost therapeutic treatments.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

Science.gov (United States)

Marlowe, Jennifer L; Akopian, Violetta; Karmali, Priya; Kornbrust, Douglas; Lockridge, Jennifer; Semple, Sean

2017-08-01

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Emerging use of gene expression microarrays in plant physiology.

Science.gov (United States)

Wullschleger, Stan D; Difazio, Stephen P

2003-01-01

Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology were selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.

An Efficient Covalent Coating on Glass Slides for Preparation of Optical Oligonucleotide Microarrays

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Atefeh Pourjahed

2013-12-01

The agarose-PLL microarrays had the highest signal (2546 and lowest background signal (205 in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes.

Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

Energy Technology Data Exchange (ETDEWEB)

Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.; West, Jason A. A.; Hux, Gary A.

2006-10-01

The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. The development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.

Microarray-based RNA profiling of breast cancer

DEFF Research Database (Denmark)

Larsen, Martin J; Thomassen, Mads; Tan, Qihua

2014-01-01

analyzed the same 234 breast cancers on two different microarray platforms. One dataset contained known batch-effects associated with the fabrication procedure used. The aim was to assess the significance of correcting for systematic batch-effects when integrating data from different platforms. We here...

Calibration and assessment of channel-specific biases in microarray data with extended dynamical range.

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Bengtsson, Henrik; Jönsson, Göran; Vallon-Christersson, Johan

2004-11-12

Non-linearities in observed log-ratios of gene expressions, also known as intensity dependent log-ratios, can often be accounted for by global biases in the two channels being compared. Any step in a microarray process may introduce such offsets and in this article we study the biases introduced by the microarray scanner and the image analysis software. By scanning the same spotted oligonucleotide microarray at different photomultiplier tube (PMT) gains, we have identified a channel-specific bias present in two-channel microarray data. For the scanners analyzed it was in the range of 15-25 (out of 65,535). The observed bias was very stable between subsequent scans of the same array although the PMT gain was greatly adjusted. This indicates that the bias does not originate from a step preceding the scanner detector parts. The bias varies slightly between arrays. When comparing estimates based on data from the same array, but from different scanners, we have found that different scanners introduce different amounts of bias. So do various image analysis methods. We propose a scanning protocol and a constrained affine model that allows us to identify and estimate the bias in each channel. Backward transformation removes the bias and brings the channels to the same scale. The result is that systematic effects such as intensity dependent log-ratios are removed, but also that signal densities become much more similar. The average scan, which has a larger dynamical range and greater signal-to-noise ratio than individual scans, can then be obtained. The study shows that microarray scanners may introduce a significant bias in each channel. Such biases have to be calibrated for, otherwise systematic effects such as intensity dependent log-ratios will be observed. The proposed scanning protocol and calibration method is simple to use and is useful for evaluating scanner biases or for obtaining calibrated measurements with extended dynamical range and better precision. The

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

Institute of Scientific and Technical Information of China (English)

Paul W Anderson; Bud C Tennant; Zhenghong Lee

2006-01-01

AIM: To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography (PET) imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma.METHODS: Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips(R). Ten genes were selected for validation using quantitative RT-PCR and literature review was made.RESULTS: Testis enhanced gene transcript (BAX Inhibitor 1), alpha-fetoprotein, isocitrate dehydrogenase 3 (NAD+) beta, acetyl-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most downregulated genes.CONCLUSION: Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

Extended analysis of benchmark datasets for Agilent two-color microarrays

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Kerr Kathleen F

2007-10-01

Full Text Available Abstract Background As part of its broad and ambitious mission, the MicroArray Quality Control (MAQC project reported the results of experiments using External RNA Controls (ERCs on five microarray platforms. For most platforms, several different methods of data processing were considered. However, there was no similar consideration of different methods for processing the data from the Agilent two-color platform. While this omission is understandable given the scale of the project, it can create the false impression that there is consensus about the best way to process Agilent two-color data. It is also important to consider whether ERCs are representative of all the probes on a microarray. Results A comparison of different methods of processing Agilent two-color data shows substantial differences among methods for low-intensity genes. The sensitivity and specificity for detecting differentially expressed genes varies substantially for different methods. Analysis also reveals that the ERCs in the MAQC data only span the upper half of the intensity range, and therefore cannot be representative of all genes on the microarray. Conclusion Although ERCs demonstrate good agreement between observed and expected log-ratios on the Agilent two-color platform, such an analysis is incomplete. Simple loess normalization outperformed data processing with Agilent's Feature Extraction software for accurate identification of differentially expressed genes. Results from studies using ERCs should not be over-generalized when ERCs are not representative of all probes on a microarray.

Emerging Use of Gene Expression Microarrays in Plant Physiology

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Stephen P. Difazio

2006-04-01

Full Text Available Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology were selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

Science.gov (United States)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving

Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms

DEFF Research Database (Denmark)

Gunnarsson, R.; Staaf, J.; Jansson, M.

2008-01-01

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K)...

Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

International Nuclear Information System (INIS)

Thomassen, Mads; Skov, Vibe; Eiriksdottir, Freyja; Tan, Qihua; Jochumsen, Kirsten; Fritzner, Niels; Brusgaard, Klaus; Dahlgaard, Jesper; Kruse, Torben A.

2006-01-01

The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips was three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

Science.gov (United States)

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Application of four dyes in gene expression analyses by microarrays

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van Schooten Frederik J

2005-07-01

Full Text Available Abstract Background DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes. Results Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation. Conclusion In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

DEFF Research Database (Denmark)

Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

2006-01-01

Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

"Harshlighting" small blemishes on microarrays

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Wittkowski Knut M

2005-03-01

Full Text Available Abstract Background Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs. Results We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Conclusion Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization.

Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

DEFF Research Database (Denmark)

Petersen, J.; Poulsen, Lena; Petronis, S.

2008-01-01

is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions......DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device...

Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

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Lanfranchi Gerolamo

2002-10-01

Full Text Available Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences. This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene, with each probe evaluated for hybridization kinetics (Tm and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41% were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39% could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

Analysis of baseline and cisplatin-inducible gene expression in Fanconi anemia cells using oligonucleotide-based microarrays

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Liu Johnson M

2002-11-01

Full Text Available Abstract Background Patients with Fanconi anemia (FA suffer from multiple defects, most notably of the hematological compartment (bone marrow failure, and susceptibility to cancer. Cells from FA patients show increased spontaneous chromosomal damage, which is aggravated by exposure to low concentrations of DNA cross-linking agents such as mitomycin C or cisplatin. Five of the identified FA proteins form a nuclear core complex. However, the molecular function of these proteins remains obscure. Methods Oligonucleotide microarrays were used to compare the expression of approximately 12,000 genes from FA cells with matched controls. Expression profiles were studied in lymphoblastoid cell lines derived from three different FA patients, one from the FA-A and two from the FA-C complementation groups. The isogenic control cell lines were obtained by either transfecting the cells with vectors expressing the complementing cDNAs or by using a spontaneous revertant cell line derived from the same patient. In addition, we analyzed expression profiles from two cell line couples at several time points after a 1-hour pulse treatment with a discriminating dose of cisplatin. Results Analysis of the expression profiles showed differences in expression of a number of genes, many of which have unknown function or are difficult to relate to the FA defect. However, from a selected number of proteins involved in cell cycle regulation, DNA repair and chromatin structure, Western blot analysis showed that p21waf1/Cip1 was significantly upregulated after low dose cisplatin treatment in FA cells specifically (as well as being expressed at elevated levels in untreated FA cells. Conclusions The observed increase in expression of p21waf1/Cip1 after treatment of FA cells with crosslinkers suggests that the sustained elevated levels of p21waf1/Cip1 in untreated FA cells detected by Western blot analysis likely reflect increased spontaneous damage in these cells.

Teolenn: an efficient and customizable workflow to design high-quality probes for microarray experiments

Science.gov (United States)

Jourdren, Laurent; Duclos, Aurélie; Brion, Christian; Portnoy, Thomas; Mathis, Hugues; Margeot, Antoine; Le Crom, Stéphane

2010-01-01

Despite the development of new high-throughput sequencing techniques, microarrays are still attractive tools to study small genome organisms, thanks to sample multiplexing and high-feature densities. However, the oligonucleotide design remains a delicate step for most users. A vast array of software is available to deal with this problem, but each program is developed with its own strategy, which makes the choice of the best solution difficult. Here we describe Teolenn, a universal probe design workflow developed with a flexible and customizable module organization allowing fixed or variable length oligonucleotide generation. In addition, our software is able to supply quality scores for each of the designed probes. In order to assess the relevance of these scores, we performed a real hybridization using a tiling array designed against the Trichoderma reesei fungus genome. We show that our scoring pipeline correlates with signal quality for 97.2% of all the designed probes, allowing for a posteriori comparisons between quality scores and signal intensities. This result is useful in discarding any bad scoring probes during the design step in order to get high-quality microarrays. Teolenn is available at http://transcriptome.ens.fr/teolenn/. PMID:20176570

Ultrasensitive electrochemical biosensor based on the oligonucleotide self-assembled monolayer-mediated immunosensing interface

Energy Technology Data Exchange (ETDEWEB)

Liu, Dengyou; Luo, Qimei [Science College of Hunan Agricultural University, Changsha 410128 (China); Deng, Fawen [The Fourth Hospital of Chansha, Changsha 410006 (China); Li, Zhen [Science College of Hunan Agricultural University, Changsha 410128 (China); Li, Benxiang, E-mail: 172170960@qq.com [Science College of Hunan Agricultural University, Changsha 410128 (China); Shen, Zhifa, E-mail: shenzhifa@wmu.edu.cn [Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035 (China)

2017-06-08

Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis. - Highlights: • An electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer (OASM). • OASM severs as a flexible monolayer to promote electron transfer and prohibits the direct interaction of proteins with gold substrate. • The electrochemical signal is efficiently enhanced and the unspecific adsorption or cross-reaction is eliminated. • Target protein can be detected down to 0.5 ng/mL, and the high detection specificity can be obtained. �

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: Implications for transcriptomics studies

NARCIS (Netherlands)

Ballerstedt, H.; Volkers, R.J.M.; Mars, A.E.; Hallsworth, J.E.; Santos, V.A.M.D.; Puchalka, J.; Duuren, J. van; Eggink, G.; Timmis, K.N.; Bont, J.A.M. de; Wery, J.

2007-01-01

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for

Application of microarray analysis on computer cluster and cloud platforms.

Science.gov (United States)

Bernau, C; Boulesteix, A-L; Knaus, J

2013-01-01

Analysis of recent high-dimensional biological data tends to be computationally intensive as many common approaches such as resampling or permutation tests require the basic statistical analysis to be repeated many times. A crucial advantage of these methods is that they can be easily parallelized due to the computational independence of the resampling or permutation iterations, which has induced many statistics departments to establish their own computer clusters. An alternative is to rent computing resources in the cloud, e.g. at Amazon Web Services. In this article we analyze whether a selection of statistical projects, recently implemented at our department, can be efficiently realized on these cloud resources. Moreover, we illustrate an opportunity to combine computer cluster and cloud resources. In order to compare the efficiency of computer cluster and cloud implementations and their respective parallelizations we use microarray analysis procedures and compare their runtimes on the different platforms. Amazon Web Services provide various instance types which meet the particular needs of the different statistical projects we analyzed in this paper. Moreover, the network capacity is sufficient and the parallelization is comparable in efficiency to standard computer cluster implementations. Our results suggest that many statistical projects can be efficiently realized on cloud resources. It is important to mention, however, that workflows can change substantially as a result of a shift from computer cluster to cloud computing.

Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

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Carr Steven M

2007-09-01

Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

Science.gov (United States)

Tripathy, Sushant

Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

Identifying Fishes through DNA Barcodes and Microarrays.

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Marc Kochzius

2010-09-01

Full Text Available International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S, cytochrome b (cyt b, and cytochrome oxidase subunit I (COI for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90% renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

Science.gov (United States)

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Micro-Analyzer: automatic preprocessing of Affymetrix microarray data.

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Guzzi, Pietro Hiram; Cannataro, Mario

2013-08-01

A current trend in genomics is the investigation of the cell mechanism using different technologies, in order to explain the relationship among genes, molecular processes and diseases. For instance, the combined use of gene-expression arrays and genomic arrays has been demonstrated as an effective instrument in clinical practice. Consequently, in a single experiment different kind of microarrays may be used, resulting in the production of different types of binary data (images and textual raw data). The analysis of microarray data requires an initial preprocessing phase, that makes raw data suitable for use on existing analysis platforms, such as the TIGR M4 (TM4) Suite. An additional challenge to be faced by emerging data analysis platforms is the ability to treat in a combined way those different microarray formats coupled with clinical data. In fact, resulting integrated data may include both numerical and symbolic data (e.g. gene expression and SNPs regarding molecular data), as well as temporal data (e.g. the response to a drug, time to progression and survival rate), regarding clinical data. Raw data preprocessing is a crucial step in analysis but is often performed in a manual and error prone way using different software tools. Thus novel, platform independent, and possibly open source tools enabling the semi-automatic preprocessing and annotation of different microarray data are needed. The paper presents Micro-Analyzer (Microarray Analyzer), a cross-platform tool for the automatic normalization, summarization and annotation of Affymetrix gene expression and SNP binary data. It represents the evolution of the μ-CS tool, extending the preprocessing to SNP arrays that were not allowed in μ-CS. The Micro-Analyzer is provided as a Java standalone tool and enables users to read, preprocess and analyse binary microarray data (gene expression and SNPs) by invoking TM4 platform. It avoids: (i) the manual invocation of external tools (e.g. the Affymetrix Power

Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

Science.gov (United States)

Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

2017-05-01

Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

Microarray expression profiling of human dental pulp from single subject.

Science.gov (United States)

Tete, Stefano; Mastrangelo, Filiberto; Scioletti, Anna Paola; Tranasi, Michelangelo; Raicu, Florina; Paolantonio, Michele; Stuppia, Liborio; Vinci, Raffaele; Gherlone, Enrico; Ciampoli, Cristian; Sberna, Maria Teresa; Conti, Pio

2008-01-01

Microarray is a recently developed simultaneous analysis of expression patterns of thousand of genes. The aim of this research was to evaluate the expression profile of human healthy dental pulp in order to find the presence of genes activated and encoding for proteins involved in the physiological process of human dental pulp. We report data obtained by analyzing expression profiles of human tooth pulp from single subjects, using an approach based on the amplification of the total RNA. Experiments were performed on a high-density array able to analyse about 21,000 oligonucleotide sequences of about 70 bases in duplicate, using an approach based on the amplification of the total RNA from the pulp of a single tooth. Obtained data were analyzed using the S.A.M. system (Significance Analysis of Microarray) and genes were merged according to their molecular functions and biological process by the Onto-Express software. The microarray analysis revealed 362 genes with specific pulp expression. Genes showing significant high expression were classified in genes involved in tooth development, protoncogenes, genes of collagen, DNAse, Metallopeptidases and Growth factors. We report a microarray analysis, carried out by extraction of total RNA from specimens of healthy human dental pulp tissue. This approach represents a powerful tool in the study of human normal and pathological pulp, allowing minimization of the genetic variability due to the pooling of samples from different individuals.

Transcriptional profiling of endocrine cerebro-osteodysplasia using microarray and next-generation sequencing.

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Piya Lahiry

Full Text Available BACKGROUND: Transcriptome profiling of patterns of RNA expression is a powerful approach to identify networks of genes that play a role in disease. To date, most mRNA profiling of tissues has been accomplished using microarrays, but next-generation sequencing can offer a richer and more comprehensive picture. METHODOLOGY/PRINCIPAL FINDINGS: ECO is a rare multi-system developmental disorder caused by a homozygous mutation in ICK encoding intestinal cell kinase. We performed gene expression profiling using both cDNA microarrays and next-generation mRNA sequencing (mRNA-seq of skin fibroblasts from ECO-affected subjects. We then validated a subset of differentially expressed transcripts identified by each method using quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Finally, we used gene ontology (GO to identify critical pathways and processes that were abnormal according to each technical platform. Methodologically, mRNA-seq identifies a much larger number of differentially expressed genes with much better correlation to qRT-PCR results than the microarray (r² = 0.794 and 0.137, respectively. Biologically, cDNA microarray identified functional pathways focused on anatomical structure and development, while the mRNA-seq platform identified a higher proportion of genes involved in cell division and DNA replication pathways. CONCLUSIONS/SIGNIFICANCE: Transcriptome profiling with mRNA-seq had greater sensitivity, range and accuracy than the microarray. The two platforms generated different but complementary hypotheses for further evaluation.

Identification of potential biomarkers from microarray experiments using multiple criteria optimization

International Nuclear Information System (INIS)

Sánchez-Peña, Matilde L; Isaza, Clara E; Pérez-Morales, Jaileene; Rodríguez-Padilla, Cristina; Castro, José M; Cabrera-Ríos, Mauricio

2013-01-01

Microarray experiments are capable of determining the relative expression of tens of thousands of genes simultaneously, thus resulting in very large databases. The analysis of these databases and the extraction of biologically relevant knowledge from them are challenging tasks. The identification of potential cancer biomarker genes is one of the most important aims for microarray analysis and, as such, has been widely targeted in the literature. However, identifying a set of these genes consistently across different experiments, researches, microarray platforms, or cancer types is still an elusive endeavor. Besides the inherent difficulty of the large and nonconstant variability in these experiments and the incommensurability between different microarray technologies, there is the issue of the users having to adjust a series of parameters that significantly affect the outcome of the analyses and that do not have a biological or medical meaning. In this study, the identification of potential cancer biomarkers from microarray data is casted as a multiple criteria optimization (MCO) problem. The efficient solutions to this problem, found here through data envelopment analysis (DEA), are associated to genes that are proposed as potential cancer biomarkers. The method does not require any parameter adjustment by the user, and thus fosters repeatability. The approach also allows the analysis of different microarray experiments, microarray platforms, and cancer types simultaneously. The results include the analysis of three publicly available microarray databases related to cervix cancer. This study points to the feasibility of modeling the selection of potential cancer biomarkers from microarray data as an MCO problem and solve it using DEA. Using MCO entails a new optic to the identification of potential cancer biomarkers as it does not require the definition of a threshold value to establish significance for a particular gene and the selection of a normalization

Micro-array versus nano-array platforms: a comparative study for ODN detection based on SPR enhanced ellipsometry

International Nuclear Information System (INIS)

Celen, Burcu; Piskin, Erhan; Demirel, Goekhan

2011-01-01

The rapid and sensitive detection of DNA has recently attracted worldwide attention for a variety of disease diagnoses and detection of harmful bacteria in food and drink. In this paper, we carried out a comparative study based on surface plasmon resonance enhanced ellipsometry (SPREE) for the detection of oligodeoxynucleotides (ODNs) using micro- and nano-array platforms. The micro-arrayed surfaces were fabricated by a photolithography approach using different types of mask having varying size and shape. Well-ordered arrays of high aspect ratio polymeric nanotubes were also obtained using high molecular weight polystyrene (PS) and anodic aluminum oxide (AAO) membranes having 200 nm pore diameters. The SPREE sensors were then prepared by direct coupling of thiolated probe-ODNs, which contain suitable spacer arms, on gold-coated micro- and nano-arrayed surfaces. We experimentally demonstrated that, for the first time, gold-coated free standing polymeric nano-arrayed platforms can easily be produced and lead to a significant sensor sensitivity gain compared to that of the conventional SPREE surfaces of about four times. We believe that such an enhancement in sensor response could be useful for next generation sensor systems.

Micro-array versus nano-array platforms: a comparative study for ODN detection based on SPR enhanced ellipsometry

Science.gov (United States)

Celen, Burcu; Demirel, Gökhan; Piskin, Erhan

2011-04-01

The rapid and sensitive detection of DNA has recently attracted worldwide attention for a variety of disease diagnoses and detection of harmful bacteria in food and drink. In this paper, we carried out a comparative study based on surface plasmon resonance enhanced ellipsometry (SPREE) for the detection of oligodeoxynucleotides (ODNs) using micro- and nano-array platforms. The micro-arrayed surfaces were fabricated by a photolithography approach using different types of mask having varying size and shape. Well-ordered arrays of high aspect ratio polymeric nanotubes were also obtained using high molecular weight polystyrene (PS) and anodic aluminum oxide (AAO) membranes having 200 nm pore diameters. The SPREE sensors were then prepared by direct coupling of thiolated probe-ODNs, which contain suitable spacer arms, on gold-coated micro- and nano-arrayed surfaces. We experimentally demonstrated that, for the first time, gold-coated free standing polymeric nano-arrayed platforms can easily be produced and lead to a significant sensor sensitivity gain compared to that of the conventional SPREE surfaces of about four times. We believe that such an enhancement in sensor response could be useful for next generation sensor systems.

The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

Science.gov (United States)

Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

2015-01-01

Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

Science.gov (United States)

Fenart, Stéphane; Ndong, Yves-Placide Assoumou; Duarte, Jorge; Rivière, Nathalie; Wilmer, Jeroen; van Wuytswinkel, Olivier; Lucau, Anca; Cariou, Emmanuelle; Neutelings, Godfrey; Gutierrez, Laurent; Chabbert, Brigitte; Guillot, Xavier; Tavernier, Reynald; Hawkins, Simon; Thomasset, Brigitte

2010-10-21

Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties

Development and validation of a flax (Linum usitatissimum L. gene expression oligo microarray

Directory of Open Access Journals (Sweden)

Gutierrez Laurent

2010-10-01

Full Text Available Abstract Background Flax (Linum usitatissimum L. has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars and its cellulose-rich fibres (fibre-flax cultivars used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Results Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

Energy Technology Data Exchange (ETDEWEB)

Tholouli, Eleni [Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom); MacDermott, Sarah [The Medical School, The University of Manchester, Oxford Road, M13 9PT Manchester (United Kingdom); Hoyland, Judith [School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Oxford Road, M13 9PT Manchester (United Kingdom); Yin, John Liu [Department of Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom); Byers, Richard, E-mail: richard.byers@cmft.nhs.uk [School of Cancer and Enabling Sciences, Faculty of Medical and Human Sciences, The University of Manchester, Stopford Building, Oxford Road, M13 9PT Manchester (United Kingdom)

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection in archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies.

Science.gov (United States)

Parthasarathy, Narayanan; DeShazer, David; England, Marilyn; Waag, David M

2006-11-01

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.

An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

Directory of Open Access Journals (Sweden)

Laurenzi Ian J

2009-12-01

Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

Multi-platform whole-genome microarray analyses refine the epigenetic signature of breast cancer metastasis with gene expression and copy number.

Directory of Open Access Journals (Sweden)

Joseph Andrews

2010-01-01

Full Text Available We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis.We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model using Affymetrix gene expression (U133, promoter (1.0R, and SNP/CNV (SNP 6.0 microarray platforms to correlate data from gene expression, epigenetic (DNA methylation, and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo methylation with the loss (or gain of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis.Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.

Discrimination of phytoplasmas using an oligonucleotide microarray targeting rps3, rpl22, and rps19 genes

Czech Academy of Sciences Publication Activity Database

Lenz, Ondřej; Marková, J.; Sarkisova, Tatiana; Fránová, Jana; Přibylová, Jaroslava

2015-01-01

Roč. 70, January 2015 (2015), s. 47-52 ISSN 0261-2194 Institutional support: RVO:60077344 Keywords : DNA microarray * rpl22 gene * rps19 gene * rps3 gene Subject RIV: EE - Microbiology, Virology Impact factor: 1.652, year: 2015

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DEFF Research Database (Denmark)

Dufva, Martin; Petersen, Jesper; Poulsen, Lena

2009-01-01

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to f...

Aligning experimental design with bioinformatics analysis to meet discovery research objectives.

Science.gov (United States)

Kane, Michael D

2002-01-01

The utility of genomic technology and bioinformatic analytical support to provide new and needed insight into the molecular basis of disease, development, and diversity continues to grow as more research model systems and populations are investigated. Yet deriving results that meet a specific set of research objectives requires aligning or coordinating the design of the experiment, the laboratory techniques, and the data analysis. The following paragraphs describe several important interdependent factors that need to be considered to generate high quality data from the microarray platform. These factors include aligning oligonucleotide probe design with the sample labeling strategy if oligonucleotide probes are employed, recognizing that compromises are inherent in different sample procurement methods, normalizing 2-color microarray raw data, and distinguishing the difference between gene clustering and sample clustering. These factors do not represent an exhaustive list of technical variables in microarray-based research, but this list highlights those variables that span both experimental execution and data analysis. Copyright 2001 Wiley-Liss, Inc.

Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

Directory of Open Access Journals (Sweden)

Manish Biyani

2015-07-01

Full Text Available Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density, ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.

A comparative analysis of DNA barcode microarray feature size

Directory of Open Access Journals (Sweden)

Smith Andrew M

2009-10-01

Full Text Available Abstract Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density, but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO collection used for screens of pooled yeast (Saccharomyces cerevisiae deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 μm features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 μm feature size is of comparable quality to the 30 μm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density.

The Medicinal Chemistry of Therapeutic Oligonucleotides.

Science.gov (United States)

Wan, W Brad; Seth, Punit P

2016-11-10

Oligonucleotide-based therapeutics have made rapid progress in the clinic for treatment of a variety of disease indications. Unmodified oligonucleotides are polyanionic macromolecules with poor drug-like properties. Over the past two decades, medicinal chemists have identified a number of chemical modification and conjugation strategies which can improve the nuclease stability, RNA-binding affinity, and pharmacokinetic properties of oligonucleotides for therapeutic applications. In this perspective, we present a summary of the most commonly used nucleobase, sugar and backbone modification, and conjugation strategies used in oligonucleotide medicinal chemistry.

Bioinformatics and Microarray Data Analysis on the Cloud.

Science.gov (United States)

Calabrese, Barbara; Cannataro, Mario

2016-01-01

High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data.

Analysis of oligonucleotide array experiments with repeated measures using mixed models

Directory of Open Access Journals (Sweden)

Getchell Thomas V

2004-12-01

Full Text Available Abstract Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease or absence (Control of the disease, and brain regions including olfactory bulb (OB or cerebellum (CER. In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH procedure of controlling false discovery rate (FDR at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033 determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD procedure at the level of αnew to control the family-wise error rate (FWER for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

Optimal designs for one- and two-color microarrays using mixed models: a comparative evaluation of their efficiencies.

Science.gov (United States)

Lima Passos, Valéria; Tan, Frans E S; Winkens, Bjorn; Berger, Martijn P F

2009-01-01

Comparative studies between the one- and two-color microarrays provide supportive evidence for similarities of results on differential gene expression. So far, no design comparisons between the two platforms have been undertaken. With the objective of comparing optimal designs of one- and two-color microarrays in their statistical efficiencies, techniques of design optimization were applied within a mixed model framework. A- and D-optimal designs for the one- and two-color platforms were sought for a 3 x 3 factorial experiment. The results suggest that the choice of the platform will not affect the "subjects to groups" allocation, being concordant in the two designs. However, under financial constraints, the two-color arrays are expected to have a slight upper hand in terms of efficiency of model parameters estimates, once the price of arrays is more expensive than that of subjects. This statement is especially valid for microarray studies envisaging class comparisons.

Development of a genotyping microarray for Usher syndrome.

Science.gov (United States)

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner-Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva-Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-02-01

Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

SCK-CEN Genomic Platform: the microarray technology

International Nuclear Information System (INIS)

Benotmane, R.

2006-01-01

The human body contains approximately 10 14 cells, wherein each one is a nucleus. The nucleus contains 2x23 chromosomes, or two complete sets of the human genome, one set coming from the mother and the other from the father. In principle each set includes 30.000-40.000 genes. If the genome was a book, it would be twenty-three chapters, called chromosomes,each chapter with several thousand stories, called genes. Each story made up of paragraphs, called exons and introns. Each paragraph made up of 3 letter words, called codons. Each word is written with letters called bases (AGCT). But the whole is written in a single very long sentence, which is the DNA molecule or deoxy nucleic acid. The usual state of DNA is two complementary strands intertwined forming a double helix. In the cell, DNA is duplicated during each cell division to ensure the transmission of the genome to the daughter cells. For expression, the DNA is transcribed to messenger RNA. The RNA is edited and finally translated to a protein, each three bases coding for one amino acid. When the whole message is translated, the chain of amino acids folds itself up into a distinctive shape that depends on its sequence. Proteins are the effectors of the genes, and are responsible for all metabolic, hormonal and enzymatic reactions in the cells. The expressed RNA determines the amount of proteins to be produced and subsequently the desired effect (strong or weak) in the cell. The microarray technology aims at quantifying the amount of RNA present in the cell from each expressed gene, and at evaluating the changes of these amounts after exposure of the cell to toxic chemicals, ionising radiation or other stress components. The global picture of expressed genes helps to understand the affected genetic pathways in the cell at the molecular level. The microarray technology is used in the Radiobiology and Microbiology topics to study the effect of ionising radiation on human cells and mouse tissue, as well as the

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

International Nuclear Information System (INIS)

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

2007-01-01

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients

Template-Directed Ligation of Peptides to Oligonucleotides

Science.gov (United States)

Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

1996-01-01

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

Genome-wide transcription analyses in rice using tiling microarrays

DEFF Research Database (Denmark)

Li, Lei; Wang, Xiangfeng; Stolc, Viktor

2006-01-01

. We report here a full-genome transcription analysis of the indica rice subspecies using high-density oligonucleotide tiling microarrays. Our results provided expression data support for the existence of 35,970 (81.9%) annotated gene models and identified 5,464 unique transcribed intergenic regions...... that share similar compositional properties with the annotated exons and have significant homology to other plant proteins. Elucidating and mapping of all transcribed regions revealed an association between global transcription and cytological chromosome features, and an overall similarity of transcriptional......Sequencing and computational annotation revealed several features, including high gene numbers, unusual composition of the predicted genes and a large number of genes lacking homology to known genes, that distinguish the rice (Oryza sativa) genome from that of other fully sequenced model species...

Visual Analysis of DNA Microarray Data for Accurate Molecular Identification of Non-albicans Candida Isolates from Patients with Candidemia Episodes

OpenAIRE

De Luca Ferrari, Michela; Ribeiro Resende, Mariângela; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Gonoi, Tohru; Mikami, Yuzuru; Tominaga, Kenichiro; Kamei, Katsuhiko; Zaninelli Schreiber, Angelica; Trabasso, Plinio; Moretti, Maria Luiza

2013-01-01

The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

Science.gov (United States)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing

DEFF Research Database (Denmark)

Willenbrock, Hanni; Salomon, Jesper; Søkilde, Rolf

2009-01-01

Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two...... technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate...... better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification....

Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA

Directory of Open Access Journals (Sweden)

Rosenzweig Barry A

2007-09-01

Full Text Available Abstract Background The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course. Results Incubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold. Conclusion Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.

Science.gov (United States)

Martín, Verónica; Perales, Celia; Fernández-Algar, María; Dos Santos, Helena G; Garrido, Patricia; Pernas, María; Parro, Víctor; Moreno, Miguel; García-Pérez, Javier; Alcamí, José; Torán, José Luis; Abia, David; Domingo, Esteban; Briones, Carlos

2016-01-01

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.

Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

Science.gov (United States)

Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

2016-07-15

Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

BioconductorBuntu: a Linux distribution that implements a web-based DNA microarray analysis server.

Science.gov (United States)

Geeleher, Paul; Morris, Dermot; Hinde, John P; Golden, Aaron

2009-06-01

BioconductorBuntu is a custom distribution of Ubuntu Linux that automatically installs a server-side microarray processing environment, providing a user-friendly web-based GUI to many of the tools developed by the Bioconductor Project, accessible locally or across a network. System installation is via booting off a CD image or by using a Debian package provided to upgrade an existing Ubuntu installation. In its current version, several microarray analysis pipelines are supported including oligonucleotide, dual-or single-dye experiments, including post-processing with Gene Set Enrichment Analysis. BioconductorBuntu is designed to be extensible, by server-side integration of further relevant Bioconductor modules as required, facilitated by its straightforward underlying Python-based infrastructure. BioconductorBuntu offers an ideal environment for the development of processing procedures to facilitate the analysis of next-generation sequencing datasets. BioconductorBuntu is available for download under a creative commons license along with additional documentation and a tutorial from (http://bioinf.nuigalway.ie).

Development of a genotyping microarray for Usher syndrome

Science.gov (United States)

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-01-01

Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

Science.gov (United States)

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Electronic Structures of LNA Phosphorothioate Oligonucleotides

DEFF Research Database (Denmark)

Bohr, Henrik G.; Shim, Irene; Stein, Cy

2017-01-01

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces...

A hybrid approach to device integration on a genetic analysis platform

International Nuclear Information System (INIS)

Brennan, Des; Justice, John; Aherne, Margaret; Galvin, Paul; Jary, Dorothee; Kurg, Ants; Berik, Evgeny; Macek, Milan

2012-01-01

Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization. (paper)

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

Science.gov (United States)

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

Harshlight: a "corrective make-up" program for microarray chips

Directory of Open Access Journals (Sweden)

Wittkowski Knut M

2005-12-01

Full Text Available Abstract Background Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans do show similar artifacts, which might affect subsequent analysis. Although all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs, few tools are available to help with the detection of those defects. Results We develop a novel tool, Harshlight, for the automatic detection and masking of blemishes in HDONA microarray chips. Harshlight uses a combination of statistic and image processing methods to identify three different types of defects: localized blemishes affecting a few probes, diffuse defects affecting larger areas, and extended defects which may invalidate an entire chip. Conclusion We demonstrate the use of Harshlight can materially improve analysis of HDONA chips, especially for experiments with subtle changes between samples. For the widely used MAS5 algorithm, we show that compact blemishes cause an average of 8 gene expression values per chip to change by more than 50%, two of them by more than twofold; our masking algorithm restores about two thirds of this damage. Large-scale artifacts are successfully detected and eliminated.

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

Directory of Open Access Journals (Sweden)

Tomcin S

2014-11-01

Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

Global pathway analysis using DNA microarrays in skeletal muscle of women with polycystic ovary syndrome

DEFF Research Database (Denmark)

Skov, Vibe

2007-01-01

(study 1), to investigate whether pioglitazone therapy could reverse abnormalities in the transcriptional profile of muscle associated with insulin resistance in skeletal muscle of obese PCOS patients (study 2), and to develop a microarray platform for global gene expression profiling (study 3). In study...... comparable to other commercial and custom made microarrays and is a cost-effective alternative especially in larger epidemiological studies....

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Science.gov (United States)

Jaakson, K; Zernant, J; Külm, M; Hutchinson, A; Tonisson, N; Glavac, D; Ravnik-Glavac, M; Hawlina, M; Meltzer, M R; Caruso, R C; Testa, F; Maugeri, A; Hoyng, C B; Gouras, P; Simonelli, F; Lewis, R A; Lupski, J R; Cremers, F P M; Allikmets, R

2003-11-01

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research. Copyright 2003 Wiley

Carbohydrate microarrays

DEFF Research Database (Denmark)

Park, Sungjin; Gildersleeve, Jeffrey C; Blixt, Klas Ola

2012-01-01

In the last decade, carbohydrate microarrays have been core technologies for analyzing carbohydrate-mediated recognition events in a high-throughput fashion. A number of methods have been exploited for immobilizing glycans on the solid surface in a microarray format. This microarray...... of substrate specificities of glycosyltransferases. This review covers the construction of carbohydrate microarrays, detection methods of carbohydrate microarrays and their applications in biological and biomedical research....

Efficient oligonucleotide probe selection for pan-genomic tiling arrays

Directory of Open Access Journals (Sweden)

Zhang Wei

2009-09-01

Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

Applied Study on Magnetic Nanometer Beads in Preparation of Genechip Samples

Institute of Scientific and Technical Information of China (English)

陈慧; 高华方; 谢欣; 马雪梅; 杨渝珍

2004-01-01

Summary: A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up.The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.

Radio-marking and in vivo imagery of oligonucleotides

International Nuclear Information System (INIS)

Kuehnast, Bertrand

2000-01-01

This research thesis is part of activities aimed at the development of new molecules like oligonucleotides. Its first objective was the development and validation of a marking method with fluorine-18 of oligonucleotides for their in-vivo pharmacological assessment with positron emission tomography (PET). Further investigations addressed the use of iodine-125 for oligonucleotide marking purpose. This radio-marking, and in vivo and ex vivo imagery techniques are described, and their potential is highlighted for the pharmacological assessment of different oligonucleotides

Microarray Data Processing Techniques for Genome-Scale Network Inference from Large Public Repositories.

Science.gov (United States)

Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

2016-09-19

Pre-processing of microarray data is a well-studied problem. Furthermore, all popular platforms come with their own recommended best practices for differential analysis of genes. However, for genome-scale network inference using microarray data collected from large public repositories, these methods filter out a considerable number of genes. This is primarily due to the effects of aggregating a diverse array of experiments with different technical and biological scenarios. Here we introduce a pre-processing pipeline suitable for inferring genome-scale gene networks from large microarray datasets. We show that partitioning of the available microarray datasets according to biological relevance into tissue- and process-specific categories significantly extends the limits of downstream network construction. We demonstrate the effectiveness of our pre-processing pipeline by inferring genome-scale networks for the model plant Arabidopsis thaliana using two different construction methods and a collection of 11,760 Affymetrix ATH1 microarray chips. Our pre-processing pipeline and the datasets used in this paper are made available at http://alurulab.cc.gatech.edu/microarray-pp.

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

NARCIS (Netherlands)

Sontrop, H.M.J.; Moerland, P.D.; Van den Ham, R.; Reinders, M.J.T.; Verhaegh, W.F.J.

2009-01-01

Background: Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

NARCIS (Netherlands)

Sontrop, Herman M. J.; Moerland, Perry D.; van den Ham, René; Reinders, Marcel J. T.; Verhaegh, Wim F. J.

2009-01-01

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the

A Fisheye Viewer for microarray-based gene expression data.

Science.gov (United States)

Wu, Min; Thao, Cheng; Mu, Xiangming; Munson, Ethan V

2006-10-13

Microarray has been widely used to measure the relative amounts of every mRNA transcript from the genome in a single scan. Biologists have been accustomed to reading their experimental data directly from tables. However, microarray data are quite large and are stored in a series of files in a machine-readable format, so direct reading of the full data set is not feasible. The challenge is to design a user interface that allows biologists to usefully view large tables of raw microarray-based gene expression data. This paper presents one such interface--an electronic table (E-table) that uses fisheye distortion technology. The Fisheye Viewer for microarray-based gene expression data has been successfully developed to view MIAME data stored in the MAGE-ML format. The viewer can be downloaded from the project web site http://polaris.imt.uwm.edu:7777/fisheye/. The fisheye viewer was implemented in Java so that it could run on multiple platforms. We implemented the E-table by adapting JTable, a default table implementation in the Java Swing user interface library. Fisheye views use variable magnification to balance magnification for easy viewing and compression for maximizing the amount of data on the screen. This Fisheye Viewer is a lightweight but useful tool for biologists to quickly overview the raw microarray-based gene expression data in an E-table.

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

Science.gov (United States)

Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

2011-06-01

Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

Peptide-LNA oligonucleotide conjugates

DEFF Research Database (Denmark)

Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

2013-01-01

properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

Oligonucleotide-based theranostic nanoparticles in cancer therapy

Science.gov (United States)

Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

2016-01-01

Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics. PMID:27102380

[Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis].

Science.gov (United States)

Rao, J; Liu, D; Zhang, N; He, H; Ge, F; Chen, C

2014-01-01

Fusarium wilt, caused by a soilborne pathogen Fusarium oxysporum f. sp. lilii, is the major disease of lily (Lilium L.). In order to isolate the genes differentially expressed in a resistant reaction to F. oxysporum in L. regale Wilson, a cDNA library was constructed with L. regale root during F. oxysporum infection using the suppression subtractive hybridization (SSH), and a total of 585 unique expressed sequence tags (ESTs) were obtained. Furthermore, the gene expression profiles in the incompatible interaction between L. regale and F. oxysporum were revealed by oligonucleotide microarray analysis of 585 unique ESTs comparison to the compatible interaction between a susceptible Lilium Oriental Hybrid 'Siberia' and F. oxysporum. The result of expression profile analysis indicated that the genes encoding pathogenesis-related proteins (PRs), antioxidative stress enzymes, secondary metabolism enzymes, transcription factors, signal transduction proteins as well as a large number of unknown genes were involved in early defense response of L. regale to F. oxysporum infection. Moreover, the following quantitative reverse transcription PCR (QRT-PCR) analysis confirmed reliability of the oligonucleotide microarray data. In the present study, isolation of differentially expressed genes in L. regale during response to F. oxysporum helped to uncover the molecular mechanism associated with the resistance of L. regale against F. oxysporum.

Correlation test to assess low-level processing of high-density oligonucleotide microarray data

Directory of Open Access Journals (Sweden)

Bergh Jonas

2005-03-01

Full Text Available Abstract Background There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data. Results We analyzed coregulation between genes in order to detect insufficient normalization between arrays, where coregulation is measured in terms of statistical correlation. In a large collection of genes, a random pair of genes should have on average zero correlation, hence allowing a correlation test. For all data sets that we evaluated, and the three most commonly used low-level processing procedures including MAS5, RMA and MBEI, the housekeeping-gene normalization failed the test. For a real clinical data set, RMA and MBEI showed significant correlation for absent genes. We also found that a second round of normalization on the probe set level improved normalization significantly throughout. Conclusion Previous evaluation of low-level processing in the literature has been limited to artificial spike-in and mixture data sets. In the absence of a known gold-standard, the correlation criterion allows us to assess the appropriateness of low-level processing of a specific data set and the success of normalization for subsets of genes.

Nanomedicine, microarrays and their applications in clinical microbiology

Directory of Open Access Journals (Sweden)

Özcan Deveci

2010-12-01

Full Text Available Growing interest in the future medical applications of nanotechnology is leading to the emergence of a new scientific field that called as “nanomedicine”. Nanomedicine may be defined as the investigating, treating, reconstructing and controlling human biology and health at the molecular level, using engineered nanodevices and nanostructures. Microarray technology is a revolutionary tool for elucidating roles of genes in infectious diseases, shifting from traditional methods of research to integrated approaches. This technology has great potential to provide medical diagnosis, monitor treatment and help in the development of new tools for infectious disease prevention and/or management. The aim of this paper is to provide an overview of the current application of microarray platforms and nanomedicine in the study of experimental microbiology and the impact of this technology in clinical settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

Science.gov (United States)

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

Direct integration of intensity-level data from Affymetrix and Illumina microarrays improves statistical power for robust reanalysis

Directory of Open Access Journals (Sweden)

Turnbull Arran K

2012-08-01

Full Text Available Abstract Background Affymetrix GeneChips and Illumina BeadArrays are the most widely used commercial single channel gene expression microarrays. Public data repositories are an extremely valuable resource, providing array-derived gene expression measurements from many thousands of experiments. Unfortunately many of these studies are underpowered and it is desirable to improve power by combining data from more than one study; we sought to determine whether platform-specific bias precludes direct integration of probe intensity signals for combined reanalysis. Results Using Affymetrix and Illumina data from the microarray quality control project, from our own clinical samples, and from additional publicly available datasets we evaluated several approaches to directly integrate intensity level expression data from the two platforms. After mapping probe sequences to Ensembl genes we demonstrate that, ComBat and cross platform normalisation (XPN, significantly outperform mean-centering and distance-weighted discrimination (DWD in terms of minimising inter-platform variance. In particular we observed that DWD, a popular method used in a number of previous studies, removed systematic bias at the expense of genuine biological variability, potentially reducing legitimate biological differences from integrated datasets. Conclusion Normalised and batch-corrected intensity-level data from Affymetrix and Illumina microarrays can be directly combined to generate biologically meaningful results with improved statistical power for robust, integrated reanalysis.

Scaling of gene expression data allowing the comparison of different gene expression platforms

NARCIS (Netherlands)

van Ruissen, Fred; Schaaf, Gerben J.; Kool, Marcel; Baas, Frank; Ruijter, Jan M.

2008-01-01

Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce

Exploring the use of internal and externalcontrols for assessing microarray technical performance

Directory of Open Access Journals (Sweden)

Game Laurence

2010-12-01

Full Text Available Abstract Background The maturing of gene expression microarray technology and interest in the use of microarray-based applications for clinical and diagnostic applications calls for quantitative measures of quality. This manuscript presents a retrospective study characterizing several approaches to assess technical performance of microarray data measured on the Affymetrix GeneChip platform, including whole-array metrics and information from a standard mixture of external spike-in and endogenous internal controls. Spike-in controls were found to carry the same information about technical performance as whole-array metrics and endogenous "housekeeping" genes. These results support the use of spike-in controls as general tools for performance assessment across time, experimenters and array batches, suggesting that they have potential for comparison of microarray data generated across species using different technologies. Results A layered PCA modeling methodology that uses data from a number of classes of controls (spike-in hybridization, spike-in polyA+, internal RNA degradation, endogenous or "housekeeping genes" was used for the assessment of microarray data quality. The controls provide information on multiple stages of the experimental protocol (e.g., hybridization, RNA amplification. External spike-in, hybridization and RNA labeling controls provide information related to both assay and hybridization performance whereas internal endogenous controls provide quality information on the biological sample. We find that the variance of the data generated from the external and internal controls carries critical information about technical performance; the PCA dissection of this variance is consistent with whole-array quality assessment based on a number of quality assurance/quality control (QA/QC metrics. Conclusions These results provide support for the use of both external and internal RNA control data to assess the technical quality of microarray

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants

DEFF Research Database (Denmark)

J. Carmona, Santiago; Nielsen, Morten; Schafer-Nielsen, Claus

2015-01-01

, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than...

Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

Science.gov (United States)

Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

1990-11-01

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

A fisheye viewer for microarray-based gene expression data

Directory of Open Access Journals (Sweden)

Munson Ethan V

2006-10-01

Full Text Available Abstract Background Microarray has been widely used to measure the relative amounts of every mRNA transcript from the genome in a single scan. Biologists have been accustomed to reading their experimental data directly from tables. However, microarray data are quite large and are stored in a series of files in a machine-readable format, so direct reading of the full data set is not feasible. The challenge is to design a user interface that allows biologists to usefully view large tables of raw microarray-based gene expression data. This paper presents one such interface – an electronic table (E-table that uses fisheye distortion technology. Results The Fisheye Viewer for microarray-based gene expression data has been successfully developed to view MIAME data stored in the MAGE-ML format. The viewer can be downloaded from the project web site http://polaris.imt.uwm.edu:7777/fisheye/. The fisheye viewer was implemented in Java so that it could run on multiple platforms. We implemented the E-table by adapting JTable, a default table implementation in the Java Swing user interface library. Fisheye views use variable magnification to balance magnification for easy viewing and compression for maximizing the amount of data on the screen. Conclusion This Fisheye Viewer is a lightweight but useful tool for biologists to quickly overview the raw microarray-based gene expression data in an E-table.

Statistical Methods for Comparative Phenomics Using High-Throughput Phenotype Microarrays

KAUST Repository

Sturino, Joseph

2010-01-24

We propose statistical methods for comparing phenomics data generated by the Biolog Phenotype Microarray (PM) platform for high-throughput phenotyping. Instead of the routinely used visual inspection of data with no sound inferential basis, we develop two approaches. The first approach is based on quantifying the distance between mean or median curves from two treatments and then applying a permutation test; we also consider a permutation test applied to areas under mean curves. The second approach employs functional principal component analysis. Properties of the proposed methods are investigated on both simulated data and data sets from the PM platform.

A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

Directory of Open Access Journals (Sweden)

Helene Andersson-Svahn

2011-11-01

Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

Analyses of Aloe polysaccharides using carbohydrate microarray profiling

DEFF Research Database (Denmark)

Isager Ahl, Louise; Grace, Olwen M; Pedersen, Henriette Lodberg

2018-01-01

As the popularity of Aloe vera extracts continues to rise, a desire to fully understand the individual polymer components of the leaf mesophyll, their relation to one another and the effects they have on the human body are increasing. Polysaccharides present in the leaf mesophyll have been...... identified as the components responsible for the biological activities of Aloe vera, and they have been widely studied in the past decades. However, the commonly used methods do not provide the desired platform to conduct large comparative studies of polysaccharide compositions as most of them require...... a complete or near-complete fractionation of the polymers. The objective for this study was to assess whether carbohydrate microarrays could be used for the high-throughput analysis of cell wall polysaccharides in Aloe leaf mesophyll. The method we chose is known as Comprehensive Microarray Polymer Profiling...

Electronic Structures of LNA Phosphorothioate Oligonucleotides

Directory of Open Access Journals (Sweden)

Henrik G. Bohr

2017-09-01

Full Text Available Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM calculations and chromatography experiments on locked nucleic acid (LNA phosphorothioate (PS oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules. Keywords: LNA phosphorothioate, DNA/LNA oligonucleotide, diastereoisomers, Hartree-Fock calculations, iso-potential surface, anion chromatograms

Sequence-dependent theory of oligonucleotide hybridization kinetics

International Nuclear Information System (INIS)

Marimuthu, Karthikeyan; Chakrabarti, Raj

2014-01-01

A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions

GeneChip microarrays-signal intensities, RNA concentrations and probe sequences

International Nuclear Information System (INIS)

Binder, Hans; Preibisch, Stephan

2006-01-01

GeneChip microarrays consist of hundreds of thousands of oligonucleotide probes. The transformation of their signal intensities into RNA transcript concentrations requires the knowledge of the response function of the measuring device. We analysed the 'apparatus' function of perfect match (PM) and mismatched (MM) oligonucleotide probes of GeneChip microarrays after changes of the target concentration using the results of a spiked-in experiment. In agreement with previous studies we found that a competitive two-species Langmuir-adsorption model describes the probe intensities well. Each PM and MM probe is characterized by two hybridization constants which specify the propensity of the probe to bind specific and non-specific transcripts. The affinity for non-specific hybridization is on average equal for PM and MM. The purine-pyrimidine asymmetry of base pair interaction strengths, however, causes a characteristic PM-MM intensity difference, the sign of which depends on the middle base of the probe. The affinity for specific hybridization of the PM exceeds that of the MM on average by nearly one order of magnitude because the central mismatched base only weakly contributes to the stability of the probe/target duplexes. For the first time we differentiate between the free energy parameters related to the 64 possible middle-triples of DNA/RNA oligomer duplexes with a central Watson-Crick pairing and a central mismatched pairing. Both the PM and MM probes respond to the concentration of specific transcripts, which can be estimated from the PM and MM probe intensities using the Langmuir-model. The analysis of the PM-MM intensity difference provides at least no loss of accuracy and precision of the estimated concentration compared with the PM-only estimates which in turn outperform the MM-only estimates. The results show that the processing of the PM-MM intensity difference requires the consideration of a background term due to non-specific hybridization, which is

Comparison of RNA-seq and microarray-based models for clinical endpoint prediction.

Science.gov (United States)

Zhang, Wenqian; Yu, Ying; Hertwig, Falk; Thierry-Mieg, Jean; Zhang, Wenwei; Thierry-Mieg, Danielle; Wang, Jian; Furlanello, Cesare; Devanarayan, Viswanath; Cheng, Jie; Deng, Youping; Hero, Barbara; Hong, Huixiao; Jia, Meiwen; Li, Li; Lin, Simon M; Nikolsky, Yuri; Oberthuer, André; Qing, Tao; Su, Zhenqiang; Volland, Ruth; Wang, Charles; Wang, May D; Ai, Junmei; Albanese, Davide; Asgharzadeh, Shahab; Avigad, Smadar; Bao, Wenjun; Bessarabova, Marina; Brilliant, Murray H; Brors, Benedikt; Chierici, Marco; Chu, Tzu-Ming; Zhang, Jibin; Grundy, Richard G; He, Min Max; Hebbring, Scott; Kaufman, Howard L; Lababidi, Samir; Lancashire, Lee J; Li, Yan; Lu, Xin X; Luo, Heng; Ma, Xiwen; Ning, Baitang; Noguera, Rosa; Peifer, Martin; Phan, John H; Roels, Frederik; Rosswog, Carolina; Shao, Susan; Shen, Jie; Theissen, Jessica; Tonini, Gian Paolo; Vandesompele, Jo; Wu, Po-Yen; Xiao, Wenzhong; Xu, Joshua; Xu, Weihong; Xuan, Jiekun; Yang, Yong; Ye, Zhan; Dong, Zirui; Zhang, Ke K; Yin, Ye; Zhao, Chen; Zheng, Yuanting; Wolfinger, Russell D; Shi, Tieliu; Malkas, Linda H; Berthold, Frank; Wang, Jun; Tong, Weida; Shi, Leming; Peng, Zhiyu; Fischer, Matthias

2015-06-25

Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model. We generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models. We demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.

Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry.

Science.gov (United States)

Zhu, Chenggang; Zhu, Xiangdong; Landry, James P; Cui, Zhaomeng; Li, Quanfu; Dang, Yongjun; Mi, Lan; Zheng, Fengyun; Fei, Yiyan

2016-03-16

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

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Reinders Marcel JT

2009-11-01

Full Text Available Abstract Background Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear. Results We have performed a comprehensive sensitivity analysis of microarray breast cancer classification under the two types of feature variability mentioned above. We used data from six state of the art preprocessing methods, using a compendium consisting of eight diferent datasets, involving 1131 hybridizations, containing data from both one and two-color array technology. For a wide range of classifiers, we performed a joint study on performance, concordance and stability. In the stability analysis we explicitly tested classifiers for their noise tolerance by using perturbed expression profiles that are based on uncertainty information directly related to the preprocessing methods. Our results indicate that signature composition is strongly influenced by feature variability, even if the array platform and the stratification of patient samples are identical. In addition, we show that there is often a high level of discordance between individual class assignments for signatures constructed on data coming from different preprocessing schemes, even if the actual signature composition is identical

Overexpression of Periostin and Lumican in Esophageal Squamous Cell Carcinoma

International Nuclear Information System (INIS)

Kashyap, Manoj Kumar; Marimuthu, Arivusudar; Peri, Suraj; Kumar, Ghantasala S. Sameer; Jacob, Harrys K.C.; Prasad, Thottethodi Subrahmanya Keshava; Mahmood, Riaz; Kumar, K. V. Veerendra; Kumar, M. Vijaya; Meltzer, Stephen J.; Montgomery, Elizabeth A.; Kumar, Rekha V.; Pandey, Akhilesh

2010-01-01

To identify biomarkers for early detection for esophageal squamous cell carcinoma (ESCC), we previously carried out a genome-wide gene expression profiling study using an oligonucleotide microarray platform. This analysis led to identification of several transcripts that were significantly upregulated in ESCC compared to the adjacent normal epithelium. In the current study, we performed immunohistochemical analyses of protein products for two candidates genes identified from the DNA microarray analysis, periostin (POSTN) and lumican (LUM), using tissue microarrays. Increased expression of both periostin and lumican was observed in 100% of 137 different ESCC samples arrayed on tissue microarrays. Increased expression of periostin and lumican was observed in carcinoma as well as in stromal cell in the large majority of cases. These findings suggest that these candidates can be investigated in the sera of ESCC patients using ELISA or multiple reaction monitoring (MRM) type assays to further explore their utility as biomarkers

Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

OpenAIRE

Li, Robert W; Li, CongJun

2006-01-01

Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Dis...

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

Science.gov (United States)

Tang, Weizhong; Zhou, Huafu; Li, Wei

2015-01-01

To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

Discovery of a quorum sensing modulator pharmacophore by 3D small-molecule microarray screening

DEFF Research Database (Denmark)

Marsden, David M; Nicholson, Rebecca L; Skindersoe, Mette E

2010-01-01

ligand-binding domains of the LuxR homolog CarR from Erwinia carotovora subsp. carotovora. The 3D microarray platform was used to discover the biologically active chloro-pyridine pharmacophore, which was validated using a fluorometric ligand binding assay and ITC. Analogs containing the chloro...

Exploiting fluorescence for multiplex immunoassays on protein microarrays

International Nuclear Information System (INIS)

Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

2014-01-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

International Nuclear Information System (INIS)

Stephenson, J.R.; Meulen, V. ter

1982-01-01

Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T 1 oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. (Author)

Classification across gene expression microarray studies

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Kuner Ruprecht

2009-12-01

particular, the better predictive results of DV in across platform classification indicate higher robustness of the classifier when trained on single channel data and applied to gene expression ratios. Conclusions We present a systematic evaluation of strategies for the integration of independent microarray studies in a classification task. Our findings in across studies classification may guide further research aiming on the construction of more robust and reliable methods for stratification and diagnosis in clinical practice.

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

Science.gov (United States)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

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Lung, O; Furukawa-Stoffer, T; Burton Hughes, K; Pasick, J; King, D P; Hodko, D

2017-12-01

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site. © 2016 Her Majesty the Queen in Right of Canada.

Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

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Wupeng Liao

2017-01-01

Full Text Available Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF. The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO, small interfering RNA (siRNA, and microRNA (miRNA are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Energy Technology Data Exchange (ETDEWEB)

Noor, M. Omair; Hrovat, David [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Moazami-Goudarzi, Maryam [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Espie, George S. [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada)

2015-07-23

Highlights: • Solid-phase QD-FRET transduction of isothermal tHDA amplicons on paper substrates. • Ratiometric QD-FRET transduction improves assay precision and lowers the detection limit. • Zeptomole detection limit by an iPad camera after isothermal amplification. • Tunable assay sensitivity by immobilizing different amounts of QD–probe bioconjugates. - Abstract: Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

International Nuclear Information System (INIS)

Noor, M. Omair; Hrovat, David; Moazami-Goudarzi, Maryam; Espie, George S.; Krull, Ulrich J.

2015-01-01

Highlights: • Solid-phase QD-FRET transduction of isothermal tHDA amplicons on paper substrates. • Ratiometric QD-FRET transduction improves assay precision and lowers the detection limit. • Zeptomole detection limit by an iPad camera after isothermal amplification. • Tunable assay sensitivity by immobilizing different amounts of QD–probe bioconjugates. - Abstract: Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non

Development and Validation of Protein Microarray Technology for Simultaneous Inflammatory Mediator Detection in Human Sera

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Senthooran Selvarajah

2014-01-01

Full Text Available Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL- 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the “gold standard” ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays.

Gene Expression Commons: an open platform for absolute gene expression profiling.

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Jun Seita

Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

Translating microarray data for diagnostic testing in childhood leukaemia

International Nuclear Information System (INIS)

Hoffmann, Katrin; Firth, Martin J; Beesley, Alex H; Klerk, Nicholas H de; Kees, Ursula R

2006-01-01

Recent findings from microarray studies have raised the prospect of a standardized diagnostic gene expression platform to enhance accurate diagnosis and risk stratification in paediatric acute lymphoblastic leukaemia (ALL). However, the robustness as well as the format for such a diagnostic test remains to be determined. As a step towards clinical application of these findings, we have systematically analyzed a published ALL microarray data set using Robust Multi-array Analysis (RMA) and Random Forest (RF). We examined published microarray data from 104 ALL patients specimens, that represent six different subgroups defined by cytogenetic features and immunophenotypes. Using the decision-tree based supervised learning algorithm Random Forest (RF), we determined a small set of genes for optimal subgroup distinction and subsequently validated their predictive power in an independent patient cohort. We achieved very high overall ALL subgroup prediction accuracies of about 98%, and were able to verify the robustness of these genes in an independent panel of 68 specimens obtained from a different institution and processed in a different laboratory. Our study established that the selection of discriminating genes is strongly dependent on the analysis method. This may have profound implications for clinical use, particularly when the classifier is reduced to a small set of genes. We have demonstrated that as few as 26 genes yield accurate class prediction and importantly, almost 70% of these genes have not been previously identified as essential for class distinction of the six ALL subgroups. Our finding supports the feasibility of qRT-PCR technology for standardized diagnostic testing in paediatric ALL and should, in conjunction with conventional cytogenetics lead to a more accurate classification of the disease. In addition, we have demonstrated that microarray findings from one study can be confirmed in an independent study, using an entirely independent patient cohort

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

Science.gov (United States)

Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

2012-01-01

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

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Christoph Niemietz

2015-09-01

Full Text Available The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR, expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP. Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO and small interfering RNA (siRNA designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

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M J Pont

Full Text Available Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage-restricted expression as potential targets for immunotherapy of hematological cancers.

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

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Kreil David P

2008-08-01

Full Text Available Abstract Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer. To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes

Dynamic, electronically switchable surfaces for membrane protein microarrays.

Science.gov (United States)

Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

2006-02-01

Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

Energy Technology Data Exchange (ETDEWEB)

Gao, Zhong Feng; Chen, Dong Mei [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

2015-10-15

Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. - Highlights: • Conformational switch of i-motif is used for the detection of glucose and urea. • The sensor can be regenerated. • The proposed method is successfully applied in real sample assay. • Our method is label-free and inexpensive.

ArraySolver: an algorithm for colour-coded graphical display and Wilcoxon signed-rank statistics for comparing microarray gene expression data.

Science.gov (United States)

Khan, Haseeb Ahmad

2004-01-01

The massive surge in the production of microarray data poses a great challenge for proper analysis and interpretation. In recent years numerous computational tools have been developed to extract meaningful interpretation of microarray gene expression data. However, a convenient tool for two-groups comparison of microarray data is still lacking and users have to rely on commercial statistical packages that might be costly and require special skills, in addition to extra time and effort for transferring data from one platform to other. Various statistical methods, including the t-test, analysis of variance, Pearson test and Mann-Whitney U test, have been reported for comparing microarray data, whereas the utilization of the Wilcoxon signed-rank test, which is an appropriate test for two-groups comparison of gene expression data, has largely been neglected in microarray studies. The aim of this investigation was to build an integrated tool, ArraySolver, for colour-coded graphical display and comparison of gene expression data using the Wilcoxon signed-rank test. The results of software validation showed similar outputs with ArraySolver and SPSS for large datasets. Whereas the former program appeared to be more accurate for 25 or fewer pairs (n < or = 25), suggesting its potential application in analysing molecular signatures that usually contain small numbers of genes. The main advantages of ArraySolver are easy data selection, convenient report format, accurate statistics and the familiar Excel platform.

Modern methods for the synthesis of peptide-oligonucleotide conjugates

International Nuclear Information System (INIS)

Zubin, Evgenii M; Oretskaya, Tat'yana S; Romanova, Elena A

2002-01-01

The published data on the methods of chemical solution and solid-phase synthesis of peptide-oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide-oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates, viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

Maskless localized patterning of biomolecules on carbon nanotube microarray functionalized by ultrafine atmospheric pressure plasma jet using biotin-avidin system

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Abuzairi, Tomy; Okada, Mitsuru; Purnamaningsih, Retno Wigajatri; Poespawati, Nji Raden; Iwata, Futoshi; Nagatsu, Masaaki

2016-07-01

Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.

Current Knowledge on Microarray Technology - An Overview

African Journals Online (AJOL)

Erah

Review Article. Current Knowledge ... containing libraries of oligonucleotides robotically ... measurements, and averages over each oligonucleotide. ... quality of chips produced depends critically ..... Bae EK, Lee H, Lee JS, Noh EW. Isolation ...

Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

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Seyedeh Maryam Banihashemian

2013-09-01

Full Text Available UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA and cytosine-guanine 100 mer (CG-100 DNA indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

Serious limitations of the QTL/Microarray approach for QTL gene discovery

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Warden Craig H

2010-07-01

Full Text Available Abstract Background It has been proposed that the use of gene expression microarrays in nonrecombinant parental or congenic strains can accelerate the process of isolating individual genes underlying quantitative trait loci (QTL. However, the effectiveness of this approach has not been assessed. Results Thirty-seven studies that have implemented the QTL/microarray approach in rodents were reviewed. About 30% of studies showed enrichment for QTL candidates, mostly in comparisons between congenic and background strains. Three studies led to the identification of an underlying QTL gene. To complement the literature results, a microarray experiment was performed using three mouse congenic strains isolating the effects of at least 25 biometric QTL. Results show that genes in the congenic donor regions were preferentially selected. However, within donor regions, the distribution of differentially expressed genes was homogeneous once gene density was accounted for. Genes within identical-by-descent (IBD regions were less likely to be differentially expressed in chromosome 2, but not in chromosomes 11 and 17. Furthermore, expression of QTL regulated in cis (cis eQTL showed higher expression in the background genotype, which was partially explained by the presence of single nucleotide polymorphisms (SNP. Conclusions The literature shows limited successes from the QTL/microarray approach to identify QTL genes. Our own results from microarray profiling of three congenic strains revealed a strong tendency to select cis-eQTL over trans-eQTL. IBD regions had little effect on rate of differential expression, and we provide several reasons why IBD should not be used to discard eQTL candidates. In addition, mismatch probes produced false cis-eQTL that could not be completely removed with the current strains genotypes and low probe density microarrays. The reviewed studies did not account for lack of coverage from the platforms used and therefore removed genes

Fully automated parallel oligonucleotide synthesizer

Czech Academy of Sciences Publication Activity Database

Lebl, M.; Burger, Ch.; Ellman, B.; Heiner, D.; Ibrahim, G.; Jones, A.; Nibbe, M.; Thompson, J.; Mudra, Petr; Pokorný, Vít; Poncar, Pavel; Ženíšek, Karel

2001-01-01

Roč. 66, č. 8 (2001), s. 1299-1314 ISSN 0010-0765 Institutional research plan: CEZ:AV0Z4055905 Keywords : automated oligonucleotide synthesizer Subject RIV: CC - Organic Chemistry Impact factor: 0.778, year: 2001

Identification of a radiosensitivity signature using integrative metaanalysis of published microarray data for NCI-60 cancer cells

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Kim Han

2012-07-01

Full Text Available Abstract Background In the postgenome era, a prediction of response to treatment could lead to better dose selection for patients in radiotherapy. To identify a radiosensitive gene signature and elucidate related signaling pathways, four different microarray experiments were reanalyzed before radiotherapy. Results Radiosensitivity profiling data using clonogenic assay and gene expression profiling data from four published microarray platforms applied to NCI-60 cancer cell panel were used. The survival fraction at 2 Gy (SF2, range from 0 to 1 was calculated as a measure of radiosensitivity and a linear regression model was applied to identify genes or a gene set with a correlation between expression and radiosensitivity (SF2. Radiosensitivity signature genes were identified using significant analysis of microarrays (SAM and gene set analysis was performed using a global test using linear regression model. Using the radiation-related signaling pathway and identified genes, a genetic network was generated. According to SAM, 31 genes were identified as common to all the microarray platforms and therefore a common radiosensitivity signature. In gene set analysis, functions in the cell cycle, DNA replication, and cell junction, including adherence and gap junctions were related to radiosensitivity. The integrin, VEGF, MAPK, p53, JAK-STAT and Wnt signaling pathways were overrepresented in radiosensitivity. Significant genes including ACTN1, CCND1, HCLS1, ITGB5, PFN2, PTPRC, RAB13, and WAS, which are adhesion-related molecules that were identified by both SAM and gene set analysis, and showed interaction in the genetic network with the integrin signaling pathway. Conclusions Integration of four different microarray experiments and gene selection using gene set analysis discovered possible target genes and pathways relevant to radiosensitivity. Our results suggested that the identified genes are candidates for radiosensitivity biomarkers and that

Microarray-Based Gene Expression Profiling to Elucidate Effectiveness of Fermented Codonopsis lanceolata in Mice

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Woon Yong Choi

2014-04-01

Full Text Available In this study, the effect of Codonopsis lanceolata fermented by lactic acid on controlling gene expression levels related to obesity was observed in an oligonucleotide chip microarray. Among 8170 genes, 393 genes were up regulated and 760 genes were down regulated in feeding the fermented C. lanceolata (FCL. Another 374 genes were up regulated and 527 genes down regulated without feeding the sample. The genes were not affected by the FCL sample. It was interesting that among those genes, Chytochrome P450, Dmbt1, LOC76487, and thyroid hormones, etc., were mostly up or down regulated. These genes are more related to lipid synthesis. We could conclude that the FCL possibly controlled the gene expression levels related to lipid synthesis, which resulted in reducing obesity. However, more detailed protein expression experiments should be carried out.

High quality protein microarray using in situ protein purification

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Fleischmann Robert D

2009-08-01

protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays. Conclusion An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

Development and application of a microarray meter tool to optimize microarray experiments

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Rouse Richard JD

2008-07-01

Full Text Available Abstract Background Successful microarray experimentation requires a complex interplay between the slide chemistry, the printing pins, the nucleic acid probes and targets, and the hybridization milieu. Optimization of these parameters and a careful evaluation of emerging slide chemistries are a prerequisite to any large scale array fabrication effort. We have developed a 'microarray meter' tool which assesses the inherent variations associated with microarray measurement prior to embarking on large scale projects. Findings The microarray meter consists of nucleic acid targets (reference and dynamic range control and probe components. Different plate designs containing identical probe material were formulated to accommodate different robotic and pin designs. We examined the variability in probe quality and quantity (as judged by the amount of DNA printed and remaining post-hybridization using three robots equipped with capillary printing pins. Discussion The generation of microarray data with minimal variation requires consistent quality control of the (DNA microarray manufacturing and experimental processes. Spot reproducibility is a measure primarily of the variations associated with printing. The microarray meter assesses array quality by measuring the DNA content for every feature. It provides a post-hybridization analysis of array quality by scoring probe performance using three metrics, a a measure of variability in the signal intensities, b a measure of the signal dynamic range and c a measure of variability of the spot morphologies.

Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples

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Tong Weida

2010-10-01

Full Text Available Abstract Background The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer’s expiration dates. Researchers and microarray core facilities wonder whether expired microarrays are still useful for gene expression analysis. In addition, it was not clear whether the two human reference RNA samples established by the MAQC project in 2005 still maintained their transcriptome integrity over a period of four years. Experiments were conducted to answer these questions. Results Microarray data were generated in 2009 in three replicates for each of the two MAQC samples with either expired Affymetrix U133A or unexpired U133Plus2 microarrays. These results were compared with data obtained in 2005 on the U133Plus2 microarray. The percentage of overlap between the lists of differentially expressed genes (DEGs from U133Plus2 microarray data generated in 2009 and in 2005 was 97.44%. While there was some degree of fold change compression in the expired U133A microarrays, the percentage of overlap between the lists of DEGs from the expired and unexpired microarrays was as high as 96.99%. Moreover, the microarray data generated using the expired U133A microarrays in 2009 were highly concordant with microarray and TaqMan® data generated by the MAQC project in 2005. Conclusions Our results demonstrated that microarray data generated using U133A microarrays, which were more than four years past the manufacturer’s expiration date, were highly specific and consistent with those from unexpired microarrays in identifying DEGs despite some appreciable fold change compression and decrease in sensitivity. Our data also suggested that the

MiMiR: a comprehensive solution for storage, annotation and exchange of microarray data

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Rahman Fatimah

2005-11-01

Full Text Available Abstract Background The generation of large amounts of microarray data presents challenges for data collection, annotation, exchange and analysis. Although there are now widely accepted formats, minimum standards for data content and ontologies for microarray data, only a few groups are using them together to build and populate large-scale databases. Structured environments for data management are crucial for making full use of these data. Description The MiMiR database provides a comprehensive infrastructure for microarray data annotation, storage and exchange and is based on the MAGE format. MiMiR is MIAME-supportive, customised for use with data generated on the Affymetrix platform and includes a tool for data annotation using ontologies. Detailed information on the experiment, methods, reagents and signal intensity data can be captured in a systematic format. Reports screens permit the user to query the database, to view annotation on individual experiments and provide summary statistics. MiMiR has tools for automatic upload of the data from the microarray scanner and export to databases using MAGE-ML. Conclusion MiMiR facilitates microarray data management, annotation and exchange, in line with international guidelines. The database is valuable for underpinning research activities and promotes a systematic approach to data handling. Copies of MiMiR are freely available to academic groups under licence.

Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

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Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

2012-06-08

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

Effects of Atelocollagen Formulation Containing Oligonucleotide on Endothelial Permeability

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Koji Hanai

2012-01-01

Full Text Available Atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial. To date, atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models. However, the biological effects of this technology are not fully understood. In the present study, we investigated the effects of atelocollagen on endothelial paracellular barrier function. An atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells, and the change was dependent on the molecular size, structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation. An immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins. These changes were induced through p38 MAP kinase signaling. It is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties.

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

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Elzahar, N M; Magdy, N; El-Kosasy, Amira M; Bartlett, Michael G

2018-05-01

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

Microarray BASICA: Background Adjustment, Segmentation, Image Compression and Analysis of Microarray Images

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Jianping Hua

2004-01-01

Full Text Available This paper presents microarray BASICA: an integrated image processing tool for background adjustment, segmentation, image compression, and analysis of cDNA microarray images. BASICA uses a fast Mann-Whitney test-based algorithm to segment cDNA microarray images, and performs postprocessing to eliminate the segmentation irregularities. The segmentation results, along with the foreground and background intensities obtained with the background adjustment, are then used for independent compression of the foreground and background. We introduce a new distortion measurement for cDNA microarray image compression and devise a coding scheme by modifying the embedded block coding with optimized truncation (EBCOT algorithm (Taubman, 2000 to achieve optimal rate-distortion performance in lossy coding while still maintaining outstanding lossless compression performance. Experimental results show that the bit rate required to ensure sufficiently accurate gene expression measurement varies and depends on the quality of cDNA microarray images. For homogeneously hybridized cDNA microarray images, BASICA is able to provide from a bit rate as low as 5 bpp the gene expression data that are 99% in agreement with those of the original 32 bpp images.

Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

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Borsa, Baris A; Tuna, Bilge G; Hernandez, Frank J; Hernandez, Luiza I; Bayramoglu, Gulay; Arica, M Yakup; Ozalp, V Cengiz

2016-12-15

A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

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Xiaolong Wang

Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

Improved elucidation of biological processes linked to diabetic nephropathy by single probe-based microarray data analysis.

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Clemens D Cohen

Full Text Available BACKGROUND: Diabetic nephropathy (DN is a complex and chronic metabolic disease that evolves into a progressive fibrosing renal disorder. Effective transcriptomic profiling of slowly evolving disease processes such as DN can be problematic. The changes that occur are often subtle and can escape detection by conventional oligonucleotide DNA array analyses. METHODOLOGY/PRINCIPAL FINDINGS: We examined microdissected human renal tissue with or without DN using Affymetrix oligonucleotide microarrays (HG-U133A by standard Robust Multi-array Analysis (RMA. Subsequent gene ontology analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID showed limited detection of biological processes previously identified as central mechanisms in the development of DN (e.g. inflammation and angiogenesis. This apparent lack of sensitivity may be associated with the gene-oriented averaging of oligonucleotide probe signals, as this includes signals from cross-hybridizing probes and gene annotation that is based on out of date genomic data. We then examined the same CEL file data using a different methodology to determine how well it could correlate transcriptomic data with observed biology. ChipInspector (CI is based on single probe analysis and de novo gene annotation that bypasses probe set definitions. Both methods, RMA and CI, used at default settings yielded comparable numbers of differentially regulated genes. However, when verified by RT-PCR, the single probe based analysis demonstrated reduced background noise with enhanced sensitivity and fewer false positives. CONCLUSIONS/SIGNIFICANCE: Using a single probe based analysis approach with de novo gene annotation allowed an improved representation of the biological processes linked to the development and progression of DN. The improved analysis was exemplified by the detection of Wnt signaling pathway activation in DN, a process not previously reported to be involved in this disease.

Transcriptome sequencing of the Microarray Quality Control (MAQC RNA reference samples using next generation sequencing

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Thierry-Mieg Danielle

2009-06-01

Full Text Available Abstract Background Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC reference RNA samples using Roche's 454 Genome Sequencer FLX. Results We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database. Conclusion Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.

Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

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Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

2016-04-01

Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

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Shishkanova, T V; Volf, R; Krondak, M; Král, V

2007-05-15

A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

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Engelmann, Brett W

2017-01-01

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support.

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Matsuno, Yuki; Shoji, Takao; Kim, Shokaku; Chiba, Kazuhiro

2016-02-19

A straightforward method for the synthesis of oligonucleotide blocks using a Cbz-type alkyl-chain-soluble support (Z-ACSS) attached to the 3'-OH group of 3'-terminal nucleosides was developed. The Z-ACSS allowed for the preparation of fully protected deoxyribo- and ribo-oligonucleotides without chromatographic purification and released dimer- to tetramer-size oligonucleotide blocks via hydrogenation using a Pd/C catalyst without significant loss or migration of protective groups such as 5'-end 4,4'-dimethoxtrityl, 2-cyanoethyl on internucleotide bonds, or 2'-TBS.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

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Godinho, Bruno M D C; Gilbert, James W; Haraszti, Reka A; Coles, Andrew H; Biscans, Annabelle; Roux, Loic; Nikan, Mehran; Echeverria, Dimas; Hassler, Matthew; Khvorova, Anastasia

2017-12-01

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

SNPMClust: Bivariate Gaussian Genotype Clustering and Calling for Illumina Microarrays

Directory of Open Access Journals (Sweden)

Stephen W. Erickson

2016-07-01

Full Text Available SNPMClust is an R package for genotype clustering and calling with Illumina microarrays. It was originally developed for studies using the GoldenGate custom genotyping platform but can be used with other Illumina platforms, including Infinium BeadChip. The algorithm first rescales the fluorescent signal intensity data, adds empirically derived pseudo-data to minor allele genotype clusters, then uses the package mclust for bivariate Gaussian model fitting. We compared the accuracy and sensitivity of SNPMClust to that of GenCall, Illumina's proprietary algorithm, on a data set of 94 whole-genome amplified buccal (cheek swab DNA samples. These samples were genotyped on a custom panel which included 1064 SNPs for which the true genotype was known with high confidence. SNPMClust produced uniformly lower false call rates over a wide range of overall call rates.

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence.

Science.gov (United States)

Drost, Derek R; Novaes, Evandro; Boaventura-Novaes, Carolina; Benedict, Catherine I; Brown, Ryan S; Yin, Tongming; Tuskan, Gerald A; Kirst, Matias

2009-06-01

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ-synthesized microarray probes. Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa. A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.

Thermodynamics of Oligonucleotide Duplex Melting

Science.gov (United States)

Schreiber-Gosche, Sherrie; Edwards, Robert A.

2009-01-01

Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

DEFF Research Database (Denmark)

Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

2017-01-01

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

International Nuclear Information System (INIS)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-01-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

Energy Technology Data Exchange (ETDEWEB)

Katebi, Samira; Esmaeili, Abolghasem, E-mail: aesmaeili@sci.ui.ac.ir; Ghaedi, Kamran

2016-03-15

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

DEFF Research Database (Denmark)

Novak, Jaroslav P; Kim, Seon-Young; Xu, Jun

2006-01-01

BACKGROUND: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have...

Towards MRI microarrays.

Science.gov (United States)

Hall, Andrew; Mundell, Victoria J; Blanco-Andujar, Cristina; Bencsik, Martin; McHale, Glen; Newton, Michael I; Cave, Gareth W V

2010-04-14

Superparamagnetic iron oxide nanometre scale particles have been utilised as contrast agents to image staked target binding oligonucleotide arrays using MRI to correlate the signal intensity and T(2)* relaxation times in different NMR fluids.

Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

Science.gov (United States)

Mustonen, Enni-Kaisa; Palomäki, Tiina; Pasanen, Markku

2017-11-01

Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

Fibre optic microarrays.

Science.gov (United States)

Walt, David R

2010-01-01

This tutorial review describes how fibre optic microarrays can be used to create a variety of sensing and measurement systems. This review covers the basics of optical fibres and arrays, the different microarray architectures, and describes a multitude of applications. Such arrays enable multiplexed sensing for a variety of analytes including nucleic acids, vapours, and biomolecules. Polymer-coated fibre arrays can be used for measuring microscopic chemical phenomena, such as corrosion and localized release of biochemicals from cells. In addition, these microarrays can serve as a substrate for fundamental studies of single molecules and single cells. The review covers topics of interest to chemists, biologists, materials scientists, and engineers.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

Science.gov (United States)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-03-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (Prooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

International Nuclear Information System (INIS)

Pal, Sukdeb; Kim, Min Jung; Choo, Jaebum; Kang, Seong Ho; Lee, Kyeong-Hee; Song, Joon Myong

2008-01-01

A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes

Surface-enhanced Raman scattering detection of bacteria on microarrays at single cell levels using silver nanoparticles

International Nuclear Information System (INIS)

Zhou, Haibo; Yang, Danting; Mircescu, Nicoleta E.; Ivleva, Natalia P.; Schwarzmeier, Kathrin; Niessner, Reinhard; Haisch, Christoph; Wieser, Andreas; Schubert, Sören

2015-01-01

We describe a method for the synthesis of SERS-active silver nanoparticles (AgNPs) directly on the surface of bacteria (bacteria-AgNPs), specifically of E. coli cells. This straightforward strategy allows for the sensitive determination of bacteria on a microarray platform. Antibodies were used as selective receptors on the microarray surface. The Raman signal of bacteria-AgNPs is about 10 times higher than that obtained previously with microarrays based on mixing bacteria and AgNPs (bacteria+AgNPs). The optimum SERS enhancement of bacteria-AgNPs is obtained under 633-nm laser excitation, and this most likely is due to the plasmonic interaction of aggregated AgNPs. The method allows for an identification and quantification even of single E. coli bacteria. In our perception, this straightforward approach represents a most valuable tool for the detection of E. coli and, conceivably, of other bacteria, and thus has a large potential in environmental monitoring, medical diagnosis, and in food safety and quality control. (author)

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

DEFF Research Database (Denmark)

Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo

2017-01-01

by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex......, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12nm) in solution was revealed to be independent of concentration. The topological folding...

The use of oligonucleotide aptamers in cancer therapy

Directory of Open Access Journals (Sweden)

Adrian Odrzywolski

2016-05-01

Full Text Available Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers – particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.

Thermal Stability of Modified i-Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

DEFF Research Database (Denmark)

El-Sayed, Ahmed Ali; Pedersen, Erik B.; Khaireldin, Nahid Y.

2016-01-01

In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion of naphtha......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding...

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

Science.gov (United States)

Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

2016-05-11

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

Biological data warehousing system for identifying transcriptional regulatory sites from gene expressions of microarray data.

Science.gov (United States)

Tsou, Ann-Ping; Sun, Yi-Ming; Liu, Chia-Lin; Huang, Hsien-Da; Horng, Jorng-Tzong; Tsai, Meng-Feng; Liu, Baw-Juine

2006-07-01

Identification of transcriptional regulatory sites plays an important role in the investigation of gene regulation. For this propose, we designed and implemented a data warehouse to integrate multiple heterogeneous biological data sources with data types such as text-file, XML, image, MySQL database model, and Oracle database model. The utility of the biological data warehouse in predicting transcriptional regulatory sites of coregulated genes was explored using a synexpression group derived from a microarray study. Both of the binding sites of known transcription factors and predicted over-represented (OR) oligonucleotides were demonstrated for the gene group. The potential biological roles of both known nucleotides and one OR nucleotide were demonstrated using bioassays. Therefore, the results from the wet-lab experiments reinforce the power and utility of the data warehouse as an approach to the genome-wide search for important transcription regulatory elements that are the key to many complex biological systems.

Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

Directory of Open Access Journals (Sweden)

Kai Hoehlig

Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

Science.gov (United States)

Wang, Fenghua; Zhang, Hefei; Gao, Jingxia; Chen, Fengjiao; Chen, Sijie; Zhang, Cuizhen; Peng, Gang

2016-01-01

Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

Directory of Open Access Journals (Sweden)

Xi Chen

Full Text Available The prognosis of colorectal cancer (CRC stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections.

Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica.

Science.gov (United States)

Lindsey, Rebecca L; Frye, Jonathan G; Fedorka-Cray, Paula J; Meinersmann, Richard J

2011-10-01

In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.

Gaucher disease: transcriptome analyses using microarray or mRNA sequencing in a Gba1 mutant mouse model treated with velaglucerase alfa or imiglucerase.

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Nupur Dasgupta

Full Text Available Gaucher disease type 1, an inherited lysosomal storage disorder, is caused by mutations in GBA1 leading to defective glucocerebrosidase (GCase function and consequent excess accumulation of glucosylceramide/glucosylsphingosine in visceral organs. Enzyme replacement therapy (ERT with the biosimilars, imiglucerase (imig or velaglucerase alfa (vela improves/reverses the visceral disease. Comparative transcriptomic effects (microarray and mRNA-Seq of no ERT and ERT (imig or vela were done with liver, lung, and spleen from mice having Gba1 mutant alleles, termed D409V/null. Disease-related molecular effects, dynamic ranges, and sensitivities were compared between mRNA-Seq and microarrays and their respective analytic tools, i.e. Mixed Model ANOVA (microarray, and DESeq and edgeR (mRNA-Seq. While similar gene expression patterns were observed with both platforms, mRNA-Seq identified more differentially expressed genes (DEGs (∼3-fold than the microarrays. Among the three analytic tools, DESeq identified the maximum number of DEGs for all tissues and treatments. DESeq and edgeR comparisons revealed differences in DEGs identified. In 9V/null liver, spleen and lung, post-therapy transcriptomes approximated WT, were partially reverted, and had little change, respectively, and were concordant with the corresponding histological and biochemical findings. DEG overlaps were only 8-20% between mRNA-Seq and microarray, but the biological pathways were similar. Cell growth and proliferation, cell cycle, heme metabolism, and mitochondrial dysfunction were most altered with the Gaucher disease process. Imig and vela differentially affected specific disease pathways. Differential molecular responses were observed in direct transcriptome comparisons from imig- and vela-treated tissues. These results provide cross-validation for the mRNA-Seq and microarray platforms, and show differences between the molecular effects of two highly structurally similar ERT

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

Science.gov (United States)

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-11-19

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

Directory of Open Access Journals (Sweden)

Kei Kanie

2016-11-01

Full Text Available The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV, an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I, and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.

Science.gov (United States)

Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June

2013-06-14

Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

Comparative RNA-Seq and microarray analysis of gene expression changes in B-cell lymphomas of Canis familiaris.

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Marie Mooney

Full Text Available Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq appeared more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with

Dielectric properties of DNA oligonucleotides on the surface of silicon nanostructures

Energy Technology Data Exchange (ETDEWEB)

Bagraev, N. T., E-mail: bagraev@mail.ioffe.ru [St. Petersburg Polytechnic University (Russian Federation); Chernev, A. L. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation); Klyachkin, L. E. [St. Petersburg Polytechnic University (Russian Federation); Malyarenko, A. M. [Russian Academy of Sciences, Ioffe Physical–Technical Institute (Russian Federation); Emel’yanov, A. K.; Dubina, M. V. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation)

2016-10-15

Planar silicon nanostructures that are formed as a very narrow silicon quantum well confined by δ barriers heavily doped with boron are used to study the dielectric properties of DNA oligonucleotides deposited onto the surface of the nanostructures. The capacitance characteristics of the silicon nanostructures with oligonucleotides deposited onto their surface are determined by recording the local tunneling current–voltage characteristics by means of scanning tunneling microscopy. The results show the possibility of identifying the local dielectric properties of DNA oligonucleotide segments consisting of repeating G–C pairs. These properties apparently give grounds to correlate the segments with polymer molecules exhibiting the properties of multiferroics.

A random-access microarray for programmable droplet storage, retrieval and manipulation

International Nuclear Information System (INIS)

Tseng, Yi-Ming; Wang, Jhih-Jhe; Su, Yu-Chuan

2012-01-01

This article presents an integrated microfluidic system that is capable of programmably metering, entrapping, coalescing, addressably storing, retrieving and manipulating emulsion droplets. A multilayer, flexible PDMS chip with specially designed fluidic channels dynamically reconfigured by pneumatically actuated diaphragms is utilized to integrate a variety of droplet manipulation schemes. Once droplets are formed, their motions are coordinated by a 2D multiplexing scheme, which exploits the bidirectional movement of diaphragms to implement a random-access microarray. In the prototype demonstration, a PDMS molding and bonding process is used to fabricate the proposed microfluidic system. Emulsion droplets with desired volumes and compositions are produced, addressably stored, manipulated and retrieved from a 4 × 4 array, which employs just 4 (= 2 × log 2 4) control inputs for the operation. It has been demonstrated that (1) the integration of droplet manipulation and 2D multiplexing schemes can be achieved readily using bidirectional diaphragm valves, (2) multiplexing of an N × N array could be realized utilizing only 2 × log 2 N control inputs and (3) a multifunctional, random-access microarray can be accomplished employing a multilayer PDMS chip. As such, the demonstrated random-access microarray could potentially serve as a platform for continuous tracking and multistep processing of emulsion droplets, which is desired for various biological and chemical applications. (paper)

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

Science.gov (United States)

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays

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Gray Joanna

2010-11-01

Full Text Available Abstract Background We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism microarrays and DNA pooling (SNP-MaP employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations. The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes. Findings In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods. Conclusions Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.

Challenges to oligonucleotides-based therapeutics for Duchenne muscular dystrophy

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Goyenvalle Aurélie

2011-02-01

Full Text Available Abstract Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues.

Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy

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Shabanpoor, Fazel; Hammond, Suzan M; Abendroth, Frank; Hazell, Gareth; Wood, Matthew J.A.

2017-01-01

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases. PMID:28118087

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

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Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

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Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

2015-09-22

Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

Energy Technology Data Exchange (ETDEWEB)

J. Zhou; S.-K. Rhee; C. Schadt; T. Gentry; Z. He; X. Li; X. Liu; J. Liebich; S.C. Chong; L. Wu

2004-03-17

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several

Microarray-based classification of diffuse large B-cell lymphoma

DEFF Research Database (Denmark)

Poulsen, Christian Bjørn; Borup, Rehannah; Nielsen, Finn Cilius

2005-01-01

on the Affymetrix HG-U133A oligonucleotide arrays and improve the classification, we determined the expression profiles of pretreatment, diagnostic samples from 52 primary nodal DLBCL. METHODS AND RESULTS: First, three previously published gene lists were converted to the HG-U133A probe sets and used......OBJECTIVE: Hierarchical clusterings of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures have previously been used to classify DLBCL into Germinal Center B-cell (GCB) and Activated B-cell (ABC) types. To examine if it was feasible to perform a cross-platform validation...... for hierarchical clustering. In this way, three subtypes, including the GCB type (n = 20), the ABC type (n = 25) and an intermediate group, Type-3 (n = 5), were distinguished. The CD10 and Bcl-6 expression as well as t(14;18) translocation were prevalent, but not exclusive to the GCB type. By contrast, MUM1...

Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

Czech Academy of Sciences Publication Activity Database

Novák, J.P.; Kim, S.Y.; Xu, J.; Modlich, O.; Volsky, D.J.; Honys, David; Slonczewski, J.L.; Bell, D.A.; Blattner, F.R.; Blumwald, E.; Boerma, M.; Cosio, M.; Gatalica, Z.; Hajduch, M.; Hidalgo, J.; McInnes, R.R.; Miller III, M.C.; Penkowa, M.; Rolph, M.S.; Sottosanto, J.; St-Arnaud, R.; Szego, M.J.; Twell, D.; Wang, Ch.

2006-01-01

Roč. 1, č. 27 (2006), s. 1-24 ISSN 1745-6150 R&D Projects: GA ČR GA522/06/0896 Institutional research plan: CEZ:AV0Z50380511 Keywords : GENE-EXPRESSION DATA * OLIGONUCLEOTIDE ARRAY EXPERIMENTS * ESCHERICHIA-COLI K-12 Subject RIV: EB - Genetics ; Molecular Biology

Novel Biochip Platform for Nucleic Acid Analysis

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Juan J. Diaz-Mochon

2012-06-01

Full Text Available This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top “footprint” requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

Science.gov (United States)

Kupriushkin, M S; Pyshnyĭ, D V

2012-01-01

Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

Triplex-forming ability of modified oligonucleotides

DEFF Research Database (Denmark)

Højland, Torben; Babu, Bolle Ravindra; Bryld, Torsten

2007-01-01

We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA ar...

Bayesian meta-analysis models for microarray data: a comparative study

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Song Joon J

2007-03-01

Full Text Available Abstract Background With the growing abundance of microarray data, statistical methods are increasingly needed to integrate results across studies. Two common approaches for meta-analysis of microarrays include either combining gene expression measures across studies or combining summaries such as p-values, probabilities or ranks. Here, we compare two Bayesian meta-analysis models that are analogous to these methods. Results Two Bayesian meta-analysis models for microarray data have recently been introduced. The first model combines standardized gene expression measures across studies into an overall mean, accounting for inter-study variability, while the second combines probabilities of differential expression without combining expression values. Both models produce the gene-specific posterior probability of differential expression, which is the basis for inference. Since the standardized expression integration model includes inter-study variability, it may improve accuracy of results versus the probability integration model. However, due to the small number of studies typical in microarray meta-analyses, the variability between studies is challenging to estimate. The probability integration model eliminates the need to model variability between studies, and thus its implementation is more straightforward. We found in simulations of two and five studies that combining probabilities outperformed combining standardized gene expression measures for three comparison values: the percent of true discovered genes in meta-analysis versus individual studies; the percent of true genes omitted in meta-analysis versus separate studies, and the number of true discovered genes for fixed levels of Bayesian false discovery. We identified similar results when pooling two independent studies of Bacillus subtilis. We assumed that each study was produced from the same microarray platform with only two conditions: a treatment and control, and that the data sets

Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

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Ajay Kumar

2010-01-01

Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

Associating Oligonucleotides with Positively Charged Liposomes

Czech Academy of Sciences Publication Activity Database

Jurkiewicz, P.; Okruszek, A.; Hof, Martin; Langner, M.

2003-01-01

Roč. 8, č. 1 (2003), s. 77-84 ISSN 1425-8153 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : oligonucleotides * fluorescence correlation spectroscopy * DOTAP Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.455, year: 2003

THERAPEUTIC ANTISENSE OLIGONUCLEOTIDES AGAINST CANCER: HURDLING TO THE CLINIC

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Pedro Miguel Duarte Moreno

2014-10-01

Full Text Available Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen, oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention.

Science.gov (United States)

Krotz, Achim H; Mehta, Rahul C; Hardee, Gregory E

2005-02-01

Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-alpha-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage. Copyright 2004 Wiley-Liss, Inc.

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

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Lykke Pedersen

2014-01-01

Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

DEFF Research Database (Denmark)

Poulsen, Christian Bjørn; Penkowa, Milena; Borup, Rehannah

2005-01-01

Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays...... in wild-type and IL-6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post-lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with approximately 12,400 probe sets corresponding to approximately 10...... in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight...

The EADGENE Microarray Data Analysis Workshop

DEFF Research Database (Denmark)

de Koning, Dirk-Jan; Jaffrézic, Florence; Lund, Mogens Sandø

2007-01-01

Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from...... 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays...... statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful...

Identification of clinically relevant viridans streptococci by an oligonucleotide array.

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Chen, Chao Chien; Teng, Lee Jene; Kaiung, Seng; Chang, Tsung Chain

2005-04-01

Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.

Microarray-based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts.

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Liesbeth Badisco

Full Text Available Desert locusts (Schistocerca gregaria show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST database derived from S. gregaria central nervous system (CNS to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.

Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry.

Science.gov (United States)

Descamps, Emeline; Nguyen, Khoa; Bouchain-Gautier, Christelle; Filoramo, Arianna; Goux-Capes, Laurence; Goffman, Marcello; Bourgoin, Jean-Philippe; Mailley, Pascal; Livache, Thierry

2010-11-15

Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution.

A power law global error model for the identification of differentially expressed genes in microarray data

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Granucci Francesca

2004-12-01

Full Text Available Abstract Background High-density oligonucleotide microarray technology enables the discovery of genes that are transcriptionally modulated in different biological samples due to physiology, disease or intervention. Methods for the identification of these so-called "differentially expressed genes" (DEG would largely benefit from a deeper knowledge of the intrinsic measurement variability. Though it is clear that variance of repeated measures is highly dependent on the average expression level of a given gene, there is still a lack of consensus on how signal reproducibility is linked to signal intensity. The aim of this study was to empirically model the variance versus mean dependence in microarray data to improve the performance of existing methods for identifying DEG. Results In the present work we used data generated by our lab as well as publicly available data sets to show that dispersion of repeated measures depends on location of the measures themselves following a power law. This enables us to construct a power law global error model (PLGEM that is applicable to various Affymetrix GeneChip data sets. A new DEG identification method is therefore proposed, consisting of a statistic designed to make explicit use of model-derived measurement spread estimates and a resampling-based hypothesis testing algorithm. Conclusions The new method provides a control of the false positive rate, a good sensitivity vs. specificity trade-off and consistent results with varying number of replicates and even using single samples.

Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

Science.gov (United States)

Studzińska, Sylwia

2018-01-01

Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

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Mingjie Tang

Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

Science.gov (United States)

Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

2004-03-01

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

Science.gov (United States)

Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

DEFF Research Database (Denmark)

Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

2017-01-01

Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

Science.gov (United States)

Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

2017-07-15

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radioactive cDNA microarray in neurospsychiatry

International Nuclear Information System (INIS)

Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon

2003-01-01

Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

Radioactive cDNA microarray in neurospsychiatry

Energy Technology Data Exchange (ETDEWEB)

Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon [Korea University Medical School, Seoul (Korea, Republic of)

2003-02-01

Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

ArrayMining: a modular web-application for microarray analysis combining ensemble and consensus methods with cross-study normalization

Directory of Open Access Journals (Sweden)

Krasnogor Natalio

2009-10-01

Full Text Available Abstract Background Statistical analysis of DNA microarray data provides a valuable diagnostic tool for the investigation of genetic components of diseases. To take advantage of the multitude of available data sets and analysis methods, it is desirable to combine both different algorithms and data from different studies. Applying ensemble learning, consensus clustering and cross-study normalization methods for this purpose in an almost fully automated process and linking different analysis modules together under a single interface would simplify many microarray analysis tasks. Results We present ArrayMining.net, a web-application for microarray analysis that provides easy access to a wide choice of feature selection, clustering, prediction, gene set analysis and cross-study normalization methods. In contrast to other microarray-related web-tools, multiple algorithms and data sets for an analysis task can be combined using ensemble feature selection, ensemble prediction, consensus clustering and cross-platform data integration. By interlinking different analysis tools in a modular fashion, new exploratory routes become available, e.g. ensemble sample classification using features obtained from a gene set analysis and data from multiple studies. The analysis is further simplified by automatic parameter selection mechanisms and linkage to web tools and databases for functional annotation and literature mining. Conclusion ArrayMining.net is a free web-application for microarray analysis combining a broad choice of algorithms based on ensemble and consensus methods, using automatic parameter selection and integration with annotation databases.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

Science.gov (United States)

Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

2016-12-19

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides.

Science.gov (United States)

Matsuyama, Yohei; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2014-02-01

We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Recent advances in antisense oligonucleotide therapy in genetic neuromuscular diseases

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Ashok Verma

2018-01-01

Full Text Available Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Mutant genes may reduce expression of wild-type proteins, and strategies to activate expression of the wild-type proteins might provide therapeutic benefits. Also, a toxic mutant protein may cause cell death, and strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO can recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their safety and target delivery, and scientific controlled clinical trials to ascertain their therapeutic safety and efficacy have led to an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. Over the past 1 year, for the first time, the United States Food and Drug Administration has approved two ASO therapies in genetic neuromuscular diseases. This overview summarizes the recent advances in ASO technology, evolution and use of synthetic ASOs as a therapeutic platform, and the mechanism of ASO action by exon-skipping in Duchenne muscular dystrophy and exon-inclusion in spinal muscular atrophy, with comments on their advantages and limitations.

DNA microarrays : a molecular cloning manual

National Research Council Canada - National Science Library

Sambrook, Joseph; Bowtell, David

2002-01-01

.... DNA Microarrays provides authoritative, detailed instruction on the design, construction, and applications of microarrays, as well as comprehensive descriptions of the software tools and strategies...

SIGMA: A System for Integrative Genomic Microarray Analysis of Cancer Genomes

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Davies Jonathan J

2006-12-01

Full Text Available Abstract Background The prevalence of high resolution profiling of genomes has created a need for the integrative analysis of information generated from multiple methodologies and platforms. Although the majority of data in the public domain are gene expression profiles, and expression analysis software are available, the increase of array CGH studies has enabled integration of high throughput genomic and gene expression datasets. However, tools for direct mining and analysis of array CGH data are limited. Hence, there is a great need for analytical and display software tailored to cross platform integrative analysis of cancer genomes. Results We have created a user-friendly java application to facilitate sophisticated visualization and analysis such as cross-tumor and cross-platform comparisons. To demonstrate the utility of this software, we assembled array CGH data representing Affymetrix SNP chip, Stanford cDNA arrays and whole genome tiling path array platforms for cross comparison. This cancer genome database contains 267 profiles from commonly used cancer cell lines representing 14 different tissue types. Conclusion In this study we have developed an application for the visualization and analysis of data from high resolution array CGH platforms that can be adapted for analysis of multiple types of high throughput genomic datasets. Furthermore, we invite researchers using array CGH technology to deposit both their raw and processed data, as this will be a continually expanding database of cancer genomes. This publicly available resource, the System for Integrative Genomic Microarray Analysis (SIGMA of cancer genomes, can be accessed at http://sigma.bccrc.ca.

Analysis of gene expression in resynthesized Brassica napus Allopolyploids using arabidopsis 70mer oligo microarrays.

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Robert T Gaeta

Full Text Available BACKGROUND: Studies in resynthesized Brassica napus allopolyploids indicate that homoeologous chromosome exchanges in advanced generations (S(5ratio6 alter gene expression through the loss and doubling of homoeologous genes within the rearrangements. Rearrangements may also indirectly affect global gene expression if homoeologous copies of gene regulators within rearrangements have differential affects on the transcription of genes in networks. METHODOLOGY/PRINCIPAL FINDINGS: We utilized Arabidopsis 70mer oligonucleotide microarrays for exploring gene expression in three resynthesized B. napus lineages at the S(0ratio1 and S(5ratio6 generations as well as their diploid progenitors B. rapa and B. oleracea. Differential gene expression between the progenitors and additive (midparent expression in the allopolyploids were tested. The S(5ratio6 lines differed in the number of genetic rearrangements, allowing us to test if the number of genes displaying nonadditive expression was related to the number of rearrangements. Estimates using per-gene and common variance ANOVA models indicated that 6-15% of 26,107 genes were differentially expressed between the progenitors. Individual allopolyploids showed nonadditive expression for 1.6-32% of all genes. Less than 0.3% of genes displayed nonadditive expression in all S(0ratio1 lines and 0.1-0.2% were nonadditive among all S(5ratio6 lines. Differentially expressed genes in the polyploids were over-represented by genes differential between the progenitors. The total number of differentially expressed genes was correlated with the number of genetic changes in S(5ratio6 lines under the common variance model; however, there was no relationship using a per-gene variance model, and many genes showed nonadditive expression in S(0ratio1 lines. CONCLUSIONS/SIGNIFICANCE: Few genes reproducibly demonstrated nonadditive expression among lineages, suggesting few changes resulted from a general response to polyploidization

Microarray-based analysis of differential gene expression between infective and noninfective larvae of Strongyloides stercoralis.

Directory of Open Access Journals (Sweden)

Roshan Ramanathan

2011-05-01

Full Text Available Differences between noninfective first-stage (L1 and infective third-stage (L3i larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose.Oligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004, and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90. Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest.A new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and

Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

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Li CongJun

2006-09-01

Full Text Available Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Discovery Rate (FDR = 0 % were identified. The majority of these genes were repressed by butyrate and associated with cell cycle control. The expression levels of 30 selected genes identified by the microarray were confirmed using real-time PCR. The results from real-time PCR positively correlated (R = 0.867 with the results from the microarray. Conclusion This study presented the genes related to multiple signal pathways such as cell cycle control and apoptosis. The profound changes in gene expression elucidate the molecular basis for the pleiotropic effects of butyrate on biological processes. These findings enable better recognition of the full range of beneficial roles butyrate may play during cattle energy metabolism, cell growth and proliferation, and possibly in fighting gastrointestinal pathogens.

Current Knowledge on Microarray Technology - An Overview

African Journals Online (AJOL)

Erah

This paper reviews basics and updates of each microarray technology and serves to .... through protein microarrays. Protein microarrays also known as protein chips are nothing but grids that ... conditioned media, patient sera, plasma and urine. Clontech ... based antibody arrays) is similar to membrane-based antibody ...

Diagnostic and analytical applications of protein microarrays

DEFF Research Database (Denmark)

Dufva, Hans Martin; Christensen, C.B.V.

2005-01-01

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the post...

PATMA: parser of archival tissue microarray

Directory of Open Access Journals (Sweden)

Lukasz Roszkowiak

2016-12-01

Full Text Available Tissue microarrays are commonly used in modern pathology for cancer tissue evaluation, as it is a very potent technique. Tissue microarray slides are often scanned to perform computer-aided histopathological analysis of the tissue cores. For processing the image, splitting the whole virtual slide into images of individual cores is required. The only way to distinguish cores corresponding to specimens in the tissue microarray is through their arrangement. Unfortunately, distinguishing the correct order of cores is not a trivial task as they are not labelled directly on the slide. The main aim of this study was to create a procedure capable of automatically finding and extracting cores from archival images of the tissue microarrays. This software supports the work of scientists who want to perform further image processing on single cores. The proposed method is an efficient and fast procedure, working in fully automatic or semi-automatic mode. A total of 89% of punches were correctly extracted with automatic selection. With an addition of manual correction, it is possible to fully prepare the whole slide image for extraction in 2 min per tissue microarray. The proposed technique requires minimum skill and time to parse big array of cores from tissue microarray whole slide image into individual core images.

A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases.

Directory of Open Access Journals (Sweden)

Jia-Xu Chen

Full Text Available BACKGROUND: Food-borne helminthiases (FBHs have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA. The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI: 95.3-98.7% to 100.0% (95% CI: 100.0% in the protein microarray and from 97.7% (95% CI: 96.2-99.2% to 100.0% (95% CI: 100.0% in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3% to 92.1% (95% CI: 83.5-100.0% in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6% to 92.1% (95% CI: 83.5-100.0%. Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. CONCLUSIONS/SIGNIFICANCE: The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

A multilevel Lab on chip platform for DNA analysis.

Science.gov (United States)

Marasso, Simone Luigi; Giuri, Eros; Canavese, Giancarlo; Castagna, Riccardo; Quaglio, Marzia; Ferrante, Ivan; Perrone, Denis; Cocuzza, Matteo

2011-02-01

Lab-on-chips (LOCs) are critical systems that have been introduced to speed up and reduce the cost of traditional, laborious and extensive analyses in biological and biomedical fields. These ambitious and challenging issues ask for multi-disciplinary competences that range from engineering to biology. Starting from the aim to integrate microarray technology and microfluidic devices, a complex multilevel analysis platform has been designed, fabricated and tested (All rights reserved-IT Patent number TO2009A000915). This LOC successfully manages to interface microfluidic channels with standard DNA microarray glass slides, in order to implement a complete biological protocol. Typical Micro Electro Mechanical Systems (MEMS) materials and process technologies were employed. A silicon/glass microfluidic chip and a Polydimethylsiloxane (PDMS) reaction chamber were fabricated and interfaced with a standard microarray glass slide. In order to have a high disposable system all micro-elements were passive and an external apparatus provided fluidic driving and thermal control. The major microfluidic and handling problems were investigated and innovative solutions were found. Finally, an entirely automated DNA hybridization protocol was successfully tested with a significant reduction in analysis time and reagent consumption with respect to a conventional protocol.

OligoRAP - an Oligo Re-Annotation Pipeline to improve annotation and estimate target specificity

NARCIS (Netherlands)

Neerincx, P.B.T.; Rauwerda, H.; Nie, H.; Groenen, M.A.M.; Breit, T.M.; Leunissen, J.A.M.

2009-01-01

Background: High throughput gene expression studies using oligonucleotide microarrays depend on the specificity of each oligonucleotide (oligo or probe) for its target gene. However, target specific probes can only be designed when a reference genome of the species at hand were completely sequenced,

OligoRAP – an Oligo Re-Annotation Pipeline to improve annotation and estimate target specificity

NARCIS (Netherlands)

Neerincx, P.; Rauwerda, H.; Nie, H.; Groenen, M.A.M.; Breit, T.M.; Leunissen, J.A.M.

2009-01-01

Background - High throughput gene expression studies using oligonucleotide microarrays depend on the specificity of each oligonucleotide (oligo or probe) for its target gene. However, target specific probes can only be designed when a reference genome of the species at hand were completely

Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

Directory of Open Access Journals (Sweden)

Parekh-Olmedo Hetal

2006-10-01

Full Text Available Abstract Background Huntington's Disease (HD is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. Results As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP. Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene. Conclusion Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

Directory of Open Access Journals (Sweden)

Elela Sherif

2006-01-01

Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

Science.gov (United States)

Catron, Brittany; Caruso, Joseph A.; Limbach, Patrick A.

2012-06-01

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.

i-Genome: A database to summarize oligonucleotide data in genomes

Directory of Open Access Journals (Sweden)

Chang Yu-Chung

2004-10-01

Full Text Available Abstract Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

Science.gov (United States)

Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

1996-03-01

In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

Science.gov (United States)

Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

2017-12-01

Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of

Development and evaluation of new mask protocols for gene expression profiling in humans and chimpanzees

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Siegmund Kimberly D

2009-03-01

Full Text Available Abstract Background Cross-species gene expression analyses using oligonucleotide microarrays designed to evaluate a single species can provide spurious results due to mismatches between the interrogated transcriptome and arrayed probes. Based on the most recent human and chimpanzee genome assemblies, we developed updated and accessible probe masking methods that allow human Affymetrix oligonucleotide microarrays to be used for robust genome-wide expression analyses in both species. In this process, only data from oligonucleotide probes predicted to have robust hybridization sensitivity and specificity for both transcriptomes are retained for analysis. Results To characterize the utility of this resource, we applied our mask protocols to existing expression data from brains, livers, hearts, testes, and kidneys derived from both species and determined the effects probe numbers have on expression scores of specific transcripts. In all five tissues, probe sets with decreasing numbers of probes showed non-linear trends towards increased variation in expression scores. The relationships between expression variation and probe number in brain data closely matched those observed in simulated expression data sets subjected to random probe masking. However, there is evidence that additional factors affect the observed relationships between gene expression scores and probe number in tissues such as liver and kidney. In parallel, we observed that decreasing the number of probes within probe sets lead to linear increases in both gained and lost inferences of differential cross-species expression in all five tissues, which will affect the interpretation of expression data subject to masking. Conclusion We introduce a readily implemented and updated resource for human and chimpanzee transcriptome analysis through a commonly used microarray platform. Based on empirical observations derived from the analysis of five distinct data sets, we provide novel guidelines

Annotating breast cancer microarray samples using ontologies

Science.gov (United States)

Liu, Hongfang; Li, Xin; Yoon, Victoria; Clarke, Robert

2008-01-01

As the most common cancer among women, breast cancer results from the accumulation of mutations in essential genes. Recent advance in high-throughput gene expression microarray technology has inspired researchers to use the technology to assist breast cancer diagnosis, prognosis, and treatment prediction. However, the high dimensionality of microarray experiments and public access of data from many experiments have caused inconsistencies which initiated the development of controlled terminologies and ontologies for annotating microarray experiments, such as the standard microarray Gene Expression Data (MGED) ontology (MO). In this paper, we developed BCM-CO, an ontology tailored specifically for indexing clinical annotations of breast cancer microarray samples from the NCI Thesaurus. Our research showed that the coverage of NCI Thesaurus is very limited with respect to i) terms used by researchers to describe breast cancer histology (covering 22 out of 48 histology terms); ii) breast cancer cell lines (covering one out of 12 cell lines); and iii) classes corresponding to the breast cancer grading and staging. By incorporating a wider range of those terms into BCM-CO, we were able to indexed breast cancer microarray samples from GEO using BCM-CO and MGED ontology and developed a prototype system with web interface that allows the retrieval of microarray data based on the ontology annotations. PMID:18999108

Solid-phase synthesis of 2{sup '}-O-methoxyethyl oligonucleotides using dimeric phosphoramidate blocks

Energy Technology Data Exchange (ETDEWEB)

Yu, Gi Weon; Kang, Yong Han [Dept. of Applied Chemistry, Hanyang University, Ansan (Korea, Republic of)

2016-11-15

This research focused on the method of using dimeric phosphoramidite blocks to synthesize oligonucleotides for development as oligonucleotide drugs. A 16-mer oligonucleotide with the randomly selected sequence of C*C*T*C*G*C *T*C*T*C*G*C*C* C*G*C was synthesized using CC, GC, and TC dimers, a combination of monomers and dimers, or only monomers as building blocks. Using dimer blocks in this synthetic method provided a significant decrease in critical impurities that had similar properties to the main product, which was confirmed by LC-MS and HPLC analysis.

Genome-wide identification of specific oligonucleotides using artificial neural network and computational genomic analysis

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Chen Jiun-Ching

2007-05-01

Full Text Available Abstract Background Genome-wide identification of specific oligonucleotides (oligos is a computationally-intensive task and is a requirement for designing microarray probes, primers, and siRNAs. An artificial neural network (ANN is a machine learning technique that can effectively process complex and high noise data. Here, ANNs are applied to process the unique subsequence distribution for prediction of specific oligos. Results We present a novel and efficient algorithm, named the integration of ANN and BLAST (IAB algorithm, to identify specific oligos. We establish the unique marker database for human and rat gene index databases using the hash table algorithm. We then create the input vectors, via the unique marker database, to train and test the ANN. The trained ANN predicted the specific oligos with high efficiency, and these oligos were subsequently verified by BLAST. To improve the prediction performance, the ANN over-fitting issue was avoided by early stopping with the best observed error and a k-fold validation was also applied. The performance of the IAB algorithm was about 5.2, 7.1, and 6.7 times faster than the BLAST search without ANN for experimental results of 70-mer, 50-mer, and 25-mer specific oligos, respectively. In addition, the results of polymerase chain reactions showed that the primers predicted by the IAB algorithm could specifically amplify the corresponding genes. The IAB algorithm has been integrated into a previously published comprehensive web server to support microarray analysis and genome-wide iterative enrichment analysis, through which users can identify a group of desired genes and then discover the specific oligos of these genes. Conclusion The IAB algorithm has been developed to construct SpecificDB, a web server that provides a specific and valid oligo database of the probe, siRNA, and primer design for the human genome. We also demonstrate the ability of the IAB algorithm to predict specific oligos through

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

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Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

Characterization of Bovine Serum Albumin Blocking Efficiency on Epoxy-Functionalized Substrates for Microarray Applications.

Science.gov (United States)

Sun, Yung-Shin; Zhu, Xiangdong

2016-10-01

Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface. © 2015 Society for Laboratory Automation and Screening.

DNA Microarray Technology; TOPICAL

International Nuclear Information System (INIS)

WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

2002-01-01

Collaboration between Sandia National Laboratories and the University of New Mexico Biology Department resulted in the capability to train students in microarray techniques and the interpretation of data from microarray experiments. These studies provide for a better understanding of the role of stationary phase and the gene regulation involved in exit from stationary phase, which may eventually have important clinical implications. Importantly, this research trained numerous students and is the basis for three new Ph.D. projects

Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

International Nuclear Information System (INIS)

Abel, B.; Aslan, K.

2012-01-01

This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization, where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Dynamic variable selection in SNP genotype autocalling from APEX microarray data

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Zamar Ruben H

2006-11-01

Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are DNA sequence variations, occurring when a single nucleotide – adenine (A, thymine (T, cytosine (C or guanine (G – is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX. This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. Results Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU of St. Paul's Hospital (plus one negative PCR control sample. Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. Conclusion The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our

A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

Science.gov (United States)

Sachse, Konrad; Rahman, Kh Shamsur; Schnee, Christiane; Müller, Elke; Peisker, Madlen; Schumacher, Thomas; Schubert, Evelyn; Ruettger, Anke; Kaltenboeck, Bernhard; Ehricht, Ralf

2018-03-16

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Supplementing High-Density SNP Microarrays for Additional Coverage of Disease-Related Genes: Addiction as a Paradigm

Energy Technology Data Exchange (ETDEWEB)

SacconePhD, Scott F [Washington University, St. Louis; Chesler, Elissa J [ORNL; Bierut, Laura J [Washington University, St. Louis; Kalivas, Peter J [Medical College of South Carolina, Charleston; Lerman, Caryn [University of Pennsylvania; Saccone, Nancy L [Washington University, St. Louis; Uhl, George R [Johns Hopkins University; Li, Chuan-Yun [Peking University; Philip, Vivek M [ORNL; Edenberg, Howard [Indiana University; Sherry, Steven [National Center for Biotechnology Information; Feolo, Michael [National Center for Biotechnology Information; Moyzis, Robert K [Johns Hopkins University; Rutter, Joni L [National Institute of Drug Abuse

2009-01-01

Commercial SNP microarrays now provide comprehensive and affordable coverage of the human genome. However, some diseases have biologically relevant genomic regions that may require additional coverage. Addiction, for example, is thought to be influenced by complex interactions among many relevant genes and pathways. We have assembled a list of 486 biologically relevant genes nominated by a panel of experts on addiction. We then added 424 genes that showed evidence of association with addiction phenotypes through mouse QTL mappings and gene co-expression analysis. We demonstrate that there are a substantial number of SNPs in these genes that are not well represented by commercial SNP platforms. We address this problem by introducing a publicly available SNP database for addiction. The database is annotated using numeric prioritization scores indicating the extent of biological relevance. The scores incorporate a number of factors such as SNP/gene functional properties (including synonymy and promoter regions), data from mouse systems genetics and measures of human/mouse evolutionary conservation. We then used HapMap genotyping data to determine if a SNP is tagged by a commercial microarray through linkage disequilibrium. This combination of biological prioritization scores and LD tagging annotation will enable addiction researchers to supplement commercial SNP microarrays to ensure comprehensive coverage of biologically relevant regions.

MARS: Microarray analysis, retrieval, and storage system

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Scheideler Marcel

2005-04-01

Full Text Available Abstract Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS, a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at http://genome.tugraz.at.

cDNA microarray screening in food safety

International Nuclear Information System (INIS)

Roy, Sashwati; Sen, Chandan K.

2006-01-01

The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

18F-labelling of oligonucleotides using succinimido 4-[18F]fluorobenzoat

International Nuclear Information System (INIS)

Hedberg, Elisabeth; Laangstroem, Bengt

1998-01-01

A general method for the labelling of oligodeoxynucleotide and oligonucleoside phosphorothioates in the 5'-position with the positron-emitting radionuclide 18 F (t 1/2 = 110 min) is described. The label was incorporated by the reaction of succinimido 4 -[ 18 F]fluorobenzoate 4 with oligonucleotides (18- and 20-mers) modified in the 5'-position with a hexylamine linker. Oligodeoxynucleotides 5'-GCT,AAG,CGA,TGC,CTC,CGT-3' (MTCa) and 5'-GAA,CCT,CTG,AGA,GTT,CAT,CT-3' (CROa) were labelled in 20±3 % (MTCa) and 13±3 % (CROa) radiochemical yields (non-isolated, decay-corrected and based on 4). Oligonucleoside phosphorotioates MTCa (S-MTCa) and CROa (S-CROa) were labelled in 9 and 7% isolated radiochemical yield, respectively (decay-corrected and based on 4). Labelled oligonucleotides and phosphorothioate analogues were separated from their unlabelled counterparts using reversed-phase perfusion chromatography. The molecular mass of a labelled oligonucleotide CROa was determined by ESI-MS after a mixed 18 F/ 19 F fluorobenzoate labelling experiment and corresponded with the expected structure. (au)

A comprehensive platform for highly multiplexed mammalian functional genetic screens

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Cheung-Ong Kahlin

2011-05-01

Full Text Available Abstract Background Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. Results Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens. Conclusion Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray based deconvolution methods.

Design of an Enterobacteriaceae Pan-genome Microarray Chip

DEFF Research Database (Denmark)

Lukjancenko, Oksana; Ussery, David

2010-01-01

-density microarray chip has been designed, using 116 Enterobacteriaceae genome sequences, taking into account the enteric pan-genome. Probes for the microarray were checked in silico and performance of the chip, based on experimental strains from four different genera, demonstrate a relatively high ability...... to distinguish those strains on genus, species, and pathotype/serovar levels. Additionally, the microarray performed well when investigating which genes were found in a given strain of interest. The Enterobacteriaceae pan-genome microarray, based on 116 genomes, provides a valuable tool for determination...

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

Science.gov (United States)

Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

2016-04-05

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

Polyadenylation state microarray (PASTA) analysis.

Science.gov (United States)

Beilharz, Traude H; Preiss, Thomas

2011-01-01

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

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Nobumasa Hitoshi

2007-04-01

Full Text Available Abstract Background Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. Results Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. Conclusion Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross

Advanced microarray technologies for clinical diagnostics

NARCIS (Netherlands)

Pierik, Anke

2011-01-01

DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains

Highly sensitive and multiplexed platforms for allergy diagnostics

Science.gov (United States)

Monroe, Margo R.

Allergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument

Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

DEFF Research Database (Denmark)

Ries, Annika; Kumar, Rajesh; Lou, Chenguang

2016-01-01

Galactose-modified thymidine, LNA-T and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'...

Nanotechnology: moving from microarrays toward nanoarrays.

Science.gov (United States)

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

DNA Microarray Technology

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Skip to main content DNA Microarray Technology Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions Funding ...

A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus.

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Joana Teixeira de Sousa

Full Text Available The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

A cell spot microarray method for production of high density siRNA transfection microarrays

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Mpindi John-Patrick

2011-03-01

Full Text Available Abstract Background High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. Results Here, we describe the optimization of a miniaturized cell spot microarray (CSMA method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. Conclusions The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.

The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

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Ingrid E C Verhaart

2014-01-01

Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

Discovering biological progression underlying microarray samples.

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Peng Qiu

2011-04-01

Full Text Available In biological systems that undergo processes such as differentiation, a clear concept of progression exists. We present a novel computational approach, called Sample Progression Discovery (SPD, to discover patterns of biological progression underlying microarray gene expression data. SPD assumes that individual samples of a microarray dataset are related by an unknown biological process (i.e., differentiation, development, cell cycle, disease progression, and that each sample represents one unknown point along the progression of that process. SPD aims to organize the samples in a manner that reveals the underlying progression and to simultaneously identify subsets of genes that are responsible for that progression. We demonstrate the performance of SPD on a variety of microarray datasets that were generated by sampling a biological process at different points along its progression, without providing SPD any information of the underlying process. When applied to a cell cycle time series microarray dataset, SPD was not provided any prior knowledge of samples' time order or of which genes are cell-cycle regulated, yet SPD recovered the correct time order and identified many genes that have been associated with the cell cycle. When applied to B-cell differentiation data, SPD recovered the correct order of stages of normal B-cell differentiation and the linkage between preB-ALL tumor cells with their cell origin preB. When applied to mouse embryonic stem cell differentiation data, SPD uncovered a landscape of ESC differentiation into various lineages and genes that represent both generic and lineage specific processes. When applied to a prostate cancer microarray dataset, SPD identified gene modules that reflect a progression consistent with disease stages. SPD may be best viewed as a novel tool for synthesizing biological hypotheses because it provides a likely biological progression underlying a microarray dataset and, perhaps more importantly, the

Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

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Roberta D'Agata

2017-01-01

Full Text Available Gold nanoparticles (AuNPs exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

Principles of gene microarray data analysis.

Science.gov (United States)

Mocellin, Simone; Rossi, Carlo Riccardo

2007-01-01

The development of several gene expression profiling methods, such as comparative genomic hybridization (CGH), differential display, serial analysis of gene expression (SAGE), and gene microarray, together with the sequencing of the human genome, has provided an opportunity to monitor and investigate the complex cascade of molecular events leading to tumor development and progression. The availability of such large amounts of information has shifted the attention of scientists towards a nonreductionist approach to biological phenomena. High throughput technologies can be used to follow changing patterns of gene expression over time. Among them, gene microarray has become prominent because it is easier to use, does not require large-scale DNA sequencing, and allows for the parallel quantification of thousands of genes from multiple samples. Gene microarray technology is rapidly spreading worldwide and has the potential to drastically change the therapeutic approach to patients affected with tumor. Therefore, it is of paramount importance for both researchers and clinicians to know the principles underlying the analysis of the huge amount of data generated with microarray technology.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

International Nuclear Information System (INIS)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan; Durmus, Naside Gozde

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

Energy Technology Data Exchange (ETDEWEB)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan [Department of Medicine, Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Durmus, Naside Gozde, E-mail: udemirci@rics.bwh.harvard.edu [School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI (United States)

2011-09-15

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Detecting imbalanced expression of SNP alleles by minisequencing on microarrays

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Dahlgren Andreas

2004-10-01

Full Text Available Abstract Background Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system. Results The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and

Extended -Regular Sequence for Automated Analysis of Microarray Images

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Jin Hee-Jeong

2006-01-01

Full Text Available Microarray study enables us to obtain hundreds of thousands of expressions of genes or genotypes at once, and it is an indispensable technology for genome research. The first step is the analysis of scanned microarray images. This is the most important procedure for obtaining biologically reliable data. Currently most microarray image processing systems require burdensome manual block/spot indexing work. Since the amount of experimental data is increasing very quickly, automated microarray image analysis software becomes important. In this paper, we propose two automated methods for analyzing microarray images. First, we propose the extended -regular sequence to index blocks and spots, which enables a novel automatic gridding procedure. Second, we provide a methodology, hierarchical metagrid alignment, to allow reliable and efficient batch processing for a set of microarray images. Experimental results show that the proposed methods are more reliable and convenient than the commercial tools.

Cell-Based Microarrays for In Vitro Toxicology

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Wegener, Joachim

2015-07-01

DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

SLIMarray: Lightweight software for microarray facility management

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Marzolf Bruz

2006-10-01

Full Text Available Abstract Background Microarray core facilities are commonplace in biological research organizations, and need systems for accurately tracking various logistical aspects of their operation. Although these different needs could be handled separately, an integrated management system provides benefits in organization, automation and reduction in errors. Results We present SLIMarray (System for Lab Information Management of Microarrays, an open source, modular database web application capable of managing microarray inventories, sample processing and usage charges. The software allows modular configuration and is well suited for further development, providing users the flexibility to adapt it to their needs. SLIMarray Lite, a version of the software that is especially easy to install and run, is also available. Conclusion SLIMarray addresses the previously unmet need for free and open source software for managing the logistics of a microarray core facility.

Metric learning for DNA microarray data analysis

International Nuclear Information System (INIS)

Takeuchi, Ichiro; Nakagawa, Masao; Seto, Masao

2009-01-01

In many microarray studies, gene set selection is an important preliminary step for subsequent main task such as tumor classification, cancer subtype identification, etc. In this paper, we investigate the possibility of using metric learning as an alternative to gene set selection. We develop a simple metric learning algorithm aiming to use it for microarray data analysis. Exploiting a property of the algorithm, we introduce a novel approach for extending the metric learning to be adaptive. We apply the algorithm to previously studied microarray data on malignant lymphoma subtype identification.

DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY – HIGH RESOLUTION MASS SPECTROMETRY

Science.gov (United States)

Nikcevic, Irena; Wyrzykiewicz, Tadeusz K.; Limbach, Patrick A.

2010-01-01

Summary An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts

Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

Science.gov (United States)

Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

2004-11-09

We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

NARCIS (Netherlands)

Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

2015-01-01

Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that

A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

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Andrew Gehring

2014-06-01

Full Text Available Shiga toxins 1 and 2 (Stx1 and Stx2 from Shiga toxin-producing E. coli (STEC bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies and pooled horseradish peroxidase (HRP-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB, the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic and/or B-PER (a cell-disrupting, protein extraction reagent. Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

Science.gov (United States)

Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

2010-01-01

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

Probe Selection for DNA Microarrays using OligoWiz

DEFF Research Database (Denmark)

Wernersson, Rasmus; Juncker, Agnieszka; Nielsen, Henrik Bjørn

2007-01-01

Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client-server appl......Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client......-server application that offers a detailed graphical interface and real-time user interaction on the client side, and massive computer power and a large collection of species databases (400, summer 2007) on the server side. Probes are selected according to five weighted scores: cross-hybridization, deltaT(m), folding...... computer skills and can be executed from any Internet-connected computer. The probe selection procedure for a standard microarray design targeting all yeast transcripts can be completed in 1 h....

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

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Hongguang Sun

2014-01-01

Full Text Available Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

Expert Knowledge Influences Decision-Making for Couples Receiving Positive Prenatal Chromosomal Microarray Testing Results.

Science.gov (United States)

Rubel, M A; Werner-Lin, A; Barg, F K; Bernhardt, B A

2017-09-01

To assess how participants receiving abnormal prenatal genetic testing results seek information and understand the implications of results, 27 US female patients and 12 of their male partners receiving positive prenatal microarray testing results completed semi-structured phone interviews. These interviews documented participant experiences with chromosomal microarray testing, understanding of and emotional response to receiving results, factors affecting decision-making about testing and pregnancy termination, and psychosocial needs throughout the testing process. Interview data were analyzed using a modified grounded theory approach. In the absence of certainty about the implications of results, understanding of results is shaped by biomedical expert knowledge (BEK) and cultural expert knowledge (CEK). When there is a dearth of BEK, as in the case of receiving results of uncertain significance, participants rely on CEK, including religious/spiritual beliefs, "gut instinct," embodied knowledge, and social network informants. CEK is a powerful platform to guide understanding of prenatal genetic testing results. The utility of culturally situated expert knowledge during testing uncertainty emphasizes that decision-making occurs within discourses beyond the biomedical domain. These forms of "knowing" may be integrated into clinical consideration of efficacious patient assessment and counseling.

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

Science.gov (United States)

Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

1994-09-01

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

LNA-modified oligonucleotides mediate specific inhibition of microRNA function

DEFF Research Database (Denmark)

Ørom, Ulf Andersson; Kauppinen, Sakari; Lund, Anders H

2006-01-01

microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been...... observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high...... specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus...

Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

DEFF Research Database (Denmark)

Bohlin, J; Skjerve, E; Ussery, David

2008-01-01

with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies......, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore...... the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection...

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

DEFF Research Database (Denmark)

Andersson, A M; Gaardsvoll, H; Giladi, E

1990-01-01

A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA...... oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized...... the five NCAM mRNAs in rat brain....

A Java-based tool for the design of classification microarrays.

Science.gov (United States)

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for

Protein detection using biobarcodes.

Science.gov (United States)

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

Science.gov (United States)

Yokota, Toshifumi; Wood, Matthew J. A.

2013-01-01

Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

Fabrication of Biomolecule Microarrays for Cell Immobilization Using Automated Microcontact Printing.

Science.gov (United States)

Foncy, Julie; Estève, Aurore; Degache, Amélie; Colin, Camille; Cau, Jean Christophe; Malaquin, Laurent; Vieu, Christophe; Trévisiol, Emmanuelle

2018-01-01

Biomolecule microarrays are generally produced by conventional microarrayer, i.e., by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method.

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

Directory of Open Access Journals (Sweden)

Widmer Giovanni

2011-05-01

Full Text Available Abstract Background Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host. Results To investigate the magnitude of the transcriptional changes occurring during the G. lamblia life cycle we compared the transcriptome of G. lamblia trophozoites and cysts using single-color oligonucleotide microarrays. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. The comparison of the transcriptome of cysts generated in culture or extracted from feces revealed little overlap, raising the possibility of significant biological differences between the two types of cysts. Conclusions The comparison of the G. lamblia cyst and trophozoite transcriptome showed that transcripts of most genes are present at a lower level in cysts. This global view of the cyst and trophozoite transcriptome complements studies focused on the expression of selected genes during trophozoite multiplication, encystation and excystation.

Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles

DEFF Research Database (Denmark)

Cogoi, S; Jakobsen, U; Pedersen, E B

2016-01-01

KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription fa...

Shared probe design and existing microarray reanalysis using PICKY

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Chou Hui-Hsien

2010-04-01

Full Text Available Abstract Background Large genomes contain families of highly similar genes that cannot be individually identified by microarray probes. This limitation is due to thermodynamic restrictions and cannot be resolved by any computational method. Since gene annotations are updated more frequently than microarrays, another common issue facing microarray users is that existing microarrays must be routinely reanalyzed to determine probes that are still useful with respect to the updated annotations. Results PICKY 2.0 can design shared probes for sets of genes that cannot be individually identified using unique probes. PICKY 2.0 uses novel algorithms to track sharable regions among genes and to strictly distinguish them from other highly similar but nontarget regions during thermodynamic comparisons. Therefore, PICKY does not sacrifice the quality of shared probes when choosing them. The latest PICKY 2.1 includes the new capability to reanalyze existing microarray probes against updated gene sets to determine probes that are still valid to use. In addition, more precise nonlinear salt effect estimates and other improvements are added, making PICKY 2.1 more versatile to microarray users. Conclusions Shared probes allow expressed gene family members to be detected; this capability is generally more desirable than not knowing anything about these genes. Shared probes also enable the design of cross-genome microarrays, which facilitate multiple species identification in environmental samples. The new nonlinear salt effect calculation significantly increases the precision of probes at a lower buffer salt concentration, and the probe reanalysis function improves existing microarray result interpretations.

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides

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Basiri, Babak; Murph, Mandi M.; Bartlett, Michael G.

2017-08-01

Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences.

Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray

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Ballarini Annalisa

2012-07-01

Full Text Available Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT multimarker microarray (Alere Technologies GmbH, Jena, Germany, a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST were employed as reference genotyping techniques to estimate the ArrayTube resolution power. Results 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient, respectively. AT typing of this Italian collection could be easily integrated with the global P

Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

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Muyrers Joep PP

2003-01-01

Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

DEFF Research Database (Denmark)

Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

2015-01-01

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...

Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

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Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

2018-04-01

Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Integrative missing value estimation for microarray data.

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Hu, Jianjun; Li, Haifeng; Waterman, Michael S; Zhou, Xianghong Jasmine

2006-10-12

Missing value estimation is an important preprocessing step in microarray analysis. Although several methods have been developed to solve this problem, their performance is unsatisfactory for datasets with high rates of missing data, high measurement noise, or limited numbers of samples. In fact, more than 80% of the time-series datasets in Stanford Microarray Database contain less than eight samples. We present the integrative Missing Value Estimation method (iMISS) by incorporating information from multiple reference microarray datasets to improve missing value estimation. For each gene with missing data, we derive a consistent neighbor-gene list by taking reference data sets into consideration. To determine whether the given reference data sets are sufficiently informative for integration, we use a submatrix imputation approach. Our experiments showed that iMISS can significantly and consistently improve the accuracy of the state-of-the-art Local Least Square (LLS) imputation algorithm by up to 15% improvement in our benchmark tests. We demonstrated that the order-statistics-based integrative imputation algorithms can achieve significant improvements over the state-of-the-art missing value estimation approaches such as LLS and is especially good for imputing microarray datasets with a limited number of samples, high rates of missing data, or very noisy measurements. With the rapid accumulation of microarray datasets, the performance of our approach can be further improved by incorporating larger and more appropriate reference datasets.

A Java-based tool for the design of classification microarrays

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Broschat Shira L

2008-08-01

Full Text Available Abstract Background Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. Results The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. Conclusion In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays–and mixed-plasmid microarrays in particular–it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm, several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text, and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff. Weights

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

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Alsop, Eric B; Raymond, Jason

2013-01-01

Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses) for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

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Eric B Alsop

Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

Computational biology of genome expression and regulation--a review of microarray bioinformatics.

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Wang, Junbai

2008-01-01

Microarray technology is being used widely in various biomedical research areas; the corresponding microarray data analysis is an essential step toward the best utilizing of array technologies. Here we review two components of the microarray data analysis: a low level of microarray data analysis that emphasizes the designing, the quality control, and the preprocessing of microarray experiments, then a high level of microarray data analysis that focuses on the domain-specific microarray applications such as tumor classification, biomarker prediction, analyzing array CGH experiments, and reverse engineering of gene expression networks. Additionally, we will review the recent development of building a predictive model in genome expression and regulation studies. This review may help biologists grasp a basic knowledge of microarray bioinformatics as well as its potential impact on the future evolvement of biomedical research fields.

Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II; FINAL

International Nuclear Information System (INIS)

None

2000-01-01

The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank(reg s ign) finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

Platform dependence of inference on gene-wise and gene-set involvement in human lung development

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Kho Alvin T

2009-06-01

Full Text Available Abstract Background With the recent development of microarray technologies, the comparability of gene expression data obtained from different platforms poses an important problem. We evaluated two widely used platforms, Affymetrix U133 Plus 2.0 and the Illumina HumanRef-8 v2 Expression Bead Chips, for comparability in a biological system in which changes may be subtle, namely fetal lung tissue as a function of gestational age. Results We performed the comparison via sequence-based probe matching between the two platforms. "Significance grouping" was defined as a measure of comparability. Using both expression correlation and significance grouping as measures of comparability, we demonstrated that despite overall cross-platform differences at the single gene level, increased correlation between the two platforms was found in genes with higher expression level, higher probe overlap, and lower p-value. We also demonstrated that biological function as determined via KEGG pathways or GO categories is more consistent across platforms than single gene analysis. Conclusion We conclude that while the comparability of the platforms at the single gene level may be increased by increasing sample size, they are highly comparable ontologically even for subtle differences in a relatively small sample size. Biologically relevant inference should therefore be reproducible across laboratories using different platforms.

Microarray-based screening of heat shock protein inhibitors.

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Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

An oral oligonucleotide delivery system based on a thiolated polymer: Development and in vitro evaluation.

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Martien, Ronny; Hoyer, Herbert; Perera, Glen; Schnürch, Andreas Bernkop

2011-08-01

The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 μmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41% (DNase I) and 60% (intestinal fluid) were degraded in the presence of 0.02% (m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes. Copyright © 2011 Elsevier B.V. All rights reserved.

Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

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Moizza Mansoor

2008-01-01

Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy.

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Sanstead, Paul J; Stevenson, Paul; Tokmakoff, Andrei

2016-09-14

Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

Accounting for one-channel depletion improves missing value imputation in 2-dye microarray data.

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Ritz, Cecilia; Edén, Patrik

2008-01-19

For 2-dye microarray platforms, some missing values may arise from an un-measurably low RNA expression in one channel only. Information of such "one-channel depletion" is so far not included in algorithms for imputation of missing values. Calculating the mean deviation between imputed values and duplicate controls in five datasets, we show that KNN-based imputation gives a systematic bias of the imputed expression values of one-channel depleted spots. Evaluating the correction of this bias by cross-validation showed that the mean square deviation between imputed values and duplicates were reduced up to 51%, depending on dataset. By including more information in the imputation step, we more accurately estimate missing expression values.

Global transcriptional analysis of short-term hepatic stress responses in Atlantic salmon (Salmo salar) exposed to depleted uranium

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Song, You; Salbu, Brit; Teien, Hans-Christian; Heier, Lene Sørlie; Rosseland, Bjørn Olav; Høgåsen, Tore; Tollefsen, Knut Erik

2014-01-01

Potential environmental hazards of radionuclides are often studied at the individual level. Sufficient toxicogenomics data at the molecular/cellular level for understanding the effects and modes of toxic action (MoAs) of radionuclide is still lacking. The current article introduces transcriptomic data generated from a recent ecotoxicological study, with the aims to characterize the MoAs of a metallic radionuclide, deplete uranium (DU) in an ecologically and commercially important fish species, Atlantic salmon (Salmo salar). Salmon were exposed to three concentrations (0.25, 0.5 and 1.0 mg/L) of DU for 48 h. Short-term global transcriptional responses were studied using Agilent custom-designed high density 60,000-feature (60 k) salmonid oligonucleotide microarrays (oligoarray). The microarray datasets deposited at Gene Expression Omnibus (GEO ID: GSE58824) were associated with a recently published study by Song et al. (2014) in BMC Genomics. The authors describe the experimental data herein to build a platform for better understanding the toxic mechanisms and ecological hazard of radionuclides such as DU in fish. PMID:26484125

Global transcriptional analysis of short-term hepatic stress responses in Atlantic salmon (Salmo salar exposed to depleted uranium

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You Song

2014-12-01

Full Text Available Potential environmental hazards of radionuclides are often studied at the individual level. Sufficient toxicogenomics data at the molecular/cellular level for understanding the effects and modes of toxic action (MoAs of radionuclide is still lacking. The current article introduces transcriptomic data generated from a recent ecotoxicological study, with the aims to characterize the MoAs of a metallic radionuclide, deplete uranium (DU in an ecologically and commercially important fish species, Atlantic salmon (Salmo salar. Salmon were exposed to three concentrations (0.25, 0.5 and 1.0 mg/L of DU for 48 h. Short-term global transcriptional responses were studied using Agilent custom-designed high density 60,000-feature (60 k salmonid oligonucleotide microarrays (oligoarray. The microarray datasets deposited at Gene Expression Omnibus (GEO ID: GSE58824 were associated with a recently published study by Song et al. (2014 in BMC Genomics. The authors describe the experimental data herein to build a platform for better understanding the toxic mechanisms and ecological hazard of radionuclides such as DU in fish.

Spot detection and image segmentation in DNA microarray data.

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Qin, Li; Rueda, Luis; Ali, Adnan; Ngom, Alioune

2005-01-01

Following the invention of microarrays in 1994, the development and applications of this technology have grown exponentially. The numerous applications of microarray technology include clinical diagnosis and treatment, drug design and discovery, tumour detection, and environmental health research. One of the key issues in the experimental approaches utilising microarrays is to extract quantitative information from the spots, which represent genes in a given experiment. For this process, the initial stages are important and they influence future steps in the analysis. Identifying the spots and separating the background from the foreground is a fundamental problem in DNA microarray data analysis. In this review, we present an overview of state-of-the-art methods for microarray image segmentation. We discuss the foundations of the circle-shaped approach, adaptive shape segmentation, histogram-based methods and the recently introduced clustering-based techniques. We analytically show that clustering-based techniques are equivalent to the one-dimensional, standard k-means clustering algorithm that utilises the Euclidean distance.

Implementation of mutual information and bayes theorem for classification microarray data

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Dwifebri Purbolaksono, Mahendra; Widiastuti, Kurnia C.; Syahrul Mubarok, Mohamad; Adiwijaya; Aminy Ma’ruf, Firda

2018-03-01

Microarray Technology is one of technology which able to read the structure of gen. The analysis is important for this technology. It is for deciding which attribute is more important than the others. Microarray technology is able to get cancer information to diagnose a person’s gen. Preparation of microarray data is a huge problem and takes a long time. That is because microarray data contains high number of insignificant and irrelevant attributes. So, it needs a method to reduce the dimension of microarray data without eliminating important information in every attribute. This research uses Mutual Information to reduce dimension. System is built with Machine Learning approach specifically Bayes Theorem. This theorem uses a statistical and probability approach. By combining both methods, it will be powerful for Microarray Data Classification. The experiment results show that system is good to classify Microarray data with highest F1-score using Bayesian Network by 91.06%, and Naïve Bayes by 88.85%.

Universal Reference RNA as a standard for microarray experiments

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Fero Michael

2004-03-01

Full Text Available Abstract Background Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR, developed with the goal of providing hybridization signal at each microarray probe location (spot. Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. Results Human, mouse and rat URR (UHRR, UMRR and URRR, respectively were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage. Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97. Conclusion Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and

Fast and automated DNA assays on a compact disc (CD)-based microfluidic platform

Science.gov (United States)

Jia, Guangyao

Nucleic acid-based molecular diagnostics offers enormous potential for the rapid and accurate diagnosis of infectious diseases. However, most of the existing commercial tests are time-consuming and technically complicated, and are thus incompatible with the need for rapid identification of infectious agents. We have successfully developed a CD-based microfluidic platform for fast and automated DNA array hybridization and a low cost, disposable plastic microfluidic platform for polymerase chain reaction (PCR). These platforms have proved to be a promising approach to meet the requirements in terms of detection speed and operational convenience in diagnosis of infectious diseases. In the CD-based microfluidic platform for DNA hybridization, convection is introduced to the system to enhance mass transport so as to accelerate the hybridization rate since DNA hybridization is a diffusion limited reaction. Centrifugal force is utilized for sample propulsion and surface force is used for liquid gating. Standard microscope glass slides are used as the substrates for capture probes owing to their compatibility with commercially available instrumentation (e.g. laser scanners) for detection. Microfabricated polydimethylsiloxane (PDMS) structures are used to accomplish the fluidic functions required by the protocols for DNA hybridization. The assembly of the PDMS structure and the glass slide forms a flow-through hybridization unit that can be accommodated onto the CD platform for reagent manipulation. The above scheme has been validated with oligonucleotides as the targets using commercially available enzyme-labeled fluorescence (ELF 97) for detection of the hybridization events, and tested with amplicons of genomic staphylococcus DNA labeled with Cy dye. In both experiments, significantly higher fluorescence intensities were observed in the flow-through hybridization unit compared to the passive assays. The CD fluidic scheme was also adapted to the immobilization of

Integrative missing value estimation for microarray data

Directory of Open Access Journals (Sweden)

Zhou Xianghong

2006-10-01

Full Text Available Abstract Background Missing value estimation is an important preprocessing step in microarray analysis. Although several methods have been developed to solve this problem, their performance is unsatisfactory for datasets with high rates of missing data, high measurement noise, or limited numbers of samples. In fact, more than 80% of the time-series datasets in Stanford Microarray Database contain less than eight samples. Results We present the integrative Missing Value Estimation method (iMISS by incorporating information from multiple reference microarray datasets to improve missing value estimation. For each gene with missing data, we derive a consistent neighbor-gene list by taking reference data sets into consideration. To determine whether the given reference data sets are sufficiently informative for integration, we use a submatrix imputation approach. Our experiments showed that iMISS can significantly and consistently improve the accuracy of the state-of-the-art Local Least Square (LLS imputation algorithm by up to 15% improvement in our benchmark tests. Conclusion We demonstrated that the order-statistics-based integrative imputation algorithms can achieve significant improvements over the state-of-the-art missing value estimation approaches such as LLS and is especially good for imputing microarray datasets with a limited number of samples, high rates of missing data, or very noisy measurements. With the rapid accumulation of microarray datasets, the performance of our approach can be further improved by incorporating larger and more appropriate reference datasets.

Identification of mycotoxigenic fungi using an oligonucleotide microarray

CSIR Research Space (South Africa)

Barros, E

2013-01-01

Full Text Available Mycotoxins are secondary metabolites produced by fungi; they can play a role as food contaminants and have the ability to negatively influence human and animal health. To improve food safety and to protect consumers from harmful contaminants...

The molecular portraits of breast tumors are conserved across microarray platforms

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Perreard Laurent

2006-04-01

Full Text Available Abstract Background Validation of a novel gene expression signature in independent data sets is a critical step in the development of a clinically useful test for cancer patient risk-stratification. However, validation is often unconvincing because the size of the test set is typically small. To overcome this problem we used publicly available breast cancer gene expression data sets and a novel approach to data fusion, in order to validate a new breast tumor intrinsic list. Results A 105-tumor training set containing 26 sample pairs was used to derive a new breast tumor intrinsic gene list. This intrinsic list contained 1300 genes and a proliferation signature that was not present in previous breast intrinsic gene sets. We tested this list as a survival predictor on a data set of 311 tumors compiled from three independent microarray studies that were fused into a single data set using Distance Weighted Discrimination. When the new intrinsic gene set was used to hierarchically cluster this combined test set, tumors were grouped into LumA, LumB, Basal-like, HER2+/ER-, and Normal Breast-like tumor subtypes that we demonstrated in previous datasets. These subtypes were associated with significant differences in Relapse-Free and Overall Survival. Multivariate Cox analysis of the combined test set showed that the intrinsic subtype classifications added significant prognostic information that was independent of standard clinical predictors. From the combined test set, we developed an objective and unchanging classifier based upon five intrinsic subtype mean expression profiles (i.e. centroids, which is designed for single sample predictions (SSP. The SSP approach was applied to two additional independent data sets and consistently predicted survival in both systemically treated and untreated patient groups. Conclusion This study validates the "breast tumor intrinsic" subtype classification as an objective means of tumor classification that should be

Protein microarray: sensitive and effective immunodetection for drug residues

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Zer Cindy

2010-02-01

Full Text Available Abstract Background Veterinary drugs such as clenbuterol (CL and sulfamethazine (SM2 are low molecular weight ( Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g than the ci-ELISA (0.1 ng/g for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

Autoantibody profiling on human proteome microarray for biomarker discovery in cerebrospinal fluid and sera of neuropsychiatric lupus.

Directory of Open Access Journals (Sweden)

Chaojun Hu

Full Text Available Autoantibodies in cerebrospinal fluid (CSF from patients with neuropsychiatric systemic lupus erythematosus (NPSLE may be potential biomarkers for prediction, diagnosis, or prognosis of NPSLE. We used a human proteome microarray with~17,000 unique full-length human proteins to investigate autoantibodies associated with NPSLE. Twenty-nine CSF specimens from 12 NPSLE, 7 non-NPSLE, and 10 control (non-systemic lupus erythematosuspatients were screened for NPSLE-associated autoantibodies with proteome microarrays. A focused autoantigen microarray of candidate NPSLE autoantigens was applied to profile a larger cohort of CSF with patient-matched sera. We identified 137 autoantigens associated with NPSLE. Ingenuity Pathway Analysis revealed that these autoantigens were enriched for functions involved in neurological diseases (score = 43.Anti-proliferating cell nuclear antigen (PCNA was found in the CSF of NPSLE and non-NPSLE patients. The positive rates of 4 autoantibodies in CSF specimens were significantly different between the SLE (i.e., NPSLE and non-NPSLE and control groups: anti-ribosomal protein RPLP0, anti-RPLP1, anti-RPLP2, and anti-TROVE2 (also known as anti-Ro/SS-A. The positive rate for anti-SS-A associated with NPSLE was higher than that for non-NPSLE (31.11% cf. 10.71%; P = 0.045.Further analysis showed that anti-SS-A in CSF specimens was related to neuropsychiatric syndromes of the central nervous system in SLE (P = 0.009. Analysis with Spearman's rank correlation coefficient indicated that the titers of anti-RPLP2 and anti-SS-A in paired CSF and serum specimens significantly correlated. Human proteome microarrays offer a powerful platform to discover novel autoantibodies in CSF samples. Anti-SS-A autoantibodies may be potential CSF markers for NPSLE.

ValWorkBench: an open source Java library for cluster validation, with applications to microarray data analysis.

Science.gov (United States)

Giancarlo, R; Scaturro, D; Utro, F

2015-02-01

The prediction of the number of clusters in a dataset, in particular microarrays, is a fundamental task in biological data analysis, usually performed via validation measures. Unfortunately, it has received very little attention and in fact there is a growing need for software tools/libraries dedicated to it. Here we present ValWorkBench, a software library consisting of eleven well known validation measures, together with novel heuristic approximations for some of them. The main objective of this paper is to provide the interested researcher with the full software documentation of an open source cluster validation platform having the main features of being easily extendible in a homogeneous way and of offering software components that can be readily re-used. Consequently, the focus of the presentation is on the architecture of the library, since it provides an essential map that can be used to access the full software documentation, which is available at the supplementary material website [1]. The mentioned main features of ValWorkBench are also discussed and exemplified, with emphasis on software abstraction design and re-usability. A comparison with existing cluster validation software libraries, mainly in terms of the mentioned features, is also offered. It suggests that ValWorkBench is a much needed contribution to the microarray software development/algorithm engineering community. For completeness, it is important to mention that previous accurate algorithmic experimental analysis of the relative merits of each of the implemented measures [19,23,25], carried out specifically on microarray data, gives useful insights on the effectiveness of ValWorkBench for cluster validation to researchers in the microarray community interested in its use for the mentioned task. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

Energy Technology Data Exchange (ETDEWEB)

Sebastiani, F.; Comez, L.; Sacchetti, F. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); CNR, Istituto Officina dei Materiali, Unità di Perugia, c/o Dipartimento di Fisica e Geologia, Università di Perugia, 06123 Perugia (Italy); Longo, M. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); Elettra—Sincrotrone Trieste, 34149 Basovizza, Trieste (Italy); Orecchini, A.; Petrillo, C.; Paciaroni, A., E-mail: alessandro.paciaroni@fisica.unipg.it [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); De Francesco, A. [CNR-IOM OGG c/o Institut Laue-Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France); Muthmann, M. [Jülich Centre for Neutron Science, Forschungszentrum Jülich GmbH, Outstation at Heinz Maier-Leibnitz Zentrum, Lichtenbergstrasse 1, 85747 Garching (Germany); Teixeira, S. C. M. [EPSAM, Keele University, Staffordshire ST5 5BG (United Kingdom); Institut Laue–Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France)

2015-07-07

The dynamics of the human oligonucleotide AG{sub 3}(T{sub 2}AG{sub 3}){sub 3} has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2'-S-cycloadenosine.

Science.gov (United States)

Ikehara, M; Tezuka, T

1975-01-01

A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9. PMID:170595

Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

Energy Technology Data Exchange (ETDEWEB)

Awsiuk, K., E-mail: kamil.awsiuk@uj.edu.pl [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Rysz, J. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Petrou, P. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Budkowski, A. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Bernasik, A. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Kakabakos, S. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Marzec, M.M. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Raptis, I. [Institute for Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece)

2014-01-30

To immobilize effectively oligonucleotide probes on SiO{sub 2} modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m{sup 2}) and second (1.31(±0.22) mg/m{sup 2}) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m{sup 2}) and fourth (0.41(±0.11) mg/m{sup 2}) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

International Nuclear Information System (INIS)

Awsiuk, K.; Rysz, J.; Petrou, P.; Budkowski, A.; Bernasik, A.; Kakabakos, S.; Marzec, M.M.; Raptis, I.

2014-01-01

To immobilize effectively oligonucleotide probes on SiO 2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m 2 ) and second (1.31(±0.22) mg/m 2 ) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m 2 ) and fourth (0.41(±0.11) mg/m 2 ) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

DEFF Research Database (Denmark)

Taskova, Maria; Uhd, Jesper; Miotke, Laura

2017-01-01

opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

Directory of Open Access Journals (Sweden)

Mohammed Bakkali

Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

Large scale aggregate microarray analysis reveals three distinct molecular subclasses of human preeclampsia.

Science.gov (United States)

Leavey, Katherine; Bainbridge, Shannon A; Cox, Brian J

2015-01-01

Preeclampsia (PE) is a life-threatening hypertensive pathology of pregnancy affecting 3-5% of all pregnancies. To date, PE has no cure, early detection markers, or effective treatments short of the removal of what is thought to be the causative organ, the placenta, which may necessitate a preterm delivery. Additionally, numerous small placental microarray studies attempting to identify "PE-specific" genes have yielded inconsistent results. We therefore hypothesize that preeclampsia is a multifactorial disease encompassing several pathology subclasses, and that large cohort placental gene expression analysis will reveal these groups. To address our hypothesis, we utilized known bioinformatic methods to aggregate 7 microarray data sets across multiple platforms in order to generate a large data set of 173 patient samples, including 77 with preeclampsia. Unsupervised clustering of these patient samples revealed three distinct molecular subclasses of PE. This included a "canonical" PE subclass demonstrating elevated expression of known PE markers and genes associated with poor oxygenation and increased secretion, as well as two other subclasses potentially representing a poor maternal response to pregnancy and an immunological presentation of preeclampsia. Our analysis sheds new light on the heterogeneity of PE patients, and offers up additional avenues for future investigation. Hopefully, our subclassification of preeclampsia based on molecular diversity will finally lead to the development of robust diagnostics and patient-based treatments for this disorder.

Advanced spot quality analysis in two-colour microarray experiments

Directory of Open Access Journals (Sweden)

Vetter Guillaume

2008-09-01

Full Text Available Abstract Background Image analysis of microarrays and, in particular, spot quantification and spot quality control, is one of the most important steps in statistical analysis of microarray data. Recent methods of spot quality control are still in early age of development, often leading to underestimation of true positive microarray features and, consequently, to loss of important biological information. Therefore, improving and standardizing the statistical approaches of spot quality control are essential to facilitate the overall analysis of microarray data and subsequent extraction of biological information. Findings We evaluated the performance of two image analysis packages MAIA and GenePix (GP using two complementary experimental approaches with a focus on the statistical analysis of spot quality factors. First, we developed control microarrays with a priori known fluorescence ratios to verify the accuracy and precision of the ratio estimation of signal intensities. Next, we developed advanced semi-automatic protocols of spot quality evaluation in MAIA and GP and compared their performance with available facilities of spot quantitative filtering in GP. We evaluated these algorithms for standardised spot quality analysis in a whole-genome microarray experiment assessing well-characterised transcriptional modifications induced by the transcription regulator SNAI1. Using a set of RT-PCR or qRT-PCR validated microarray data, we found that the semi-automatic protocol of spot quality control we developed with MAIA allowed recovering approximately 13% more spots and 38% more differentially expressed genes (at FDR = 5% than GP with default spot filtering conditions. Conclusion Careful control of spot quality characteristics with advanced spot quality evaluation can significantly increase the amount of confident and accurate data resulting in more meaningful biological conclusions.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

Science.gov (United States)

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

Directory of Open Access Journals (Sweden)

Gil Tae Hwang

2018-01-01

Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

Directory of Open Access Journals (Sweden)

Suzan M Hammond

2014-01-01

Full Text Available Splice switching oligonucleotides (SSOs induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™” to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

An Overview of DNA Microarray Grid Alignment and Foreground Separation Approaches

Directory of Open Access Journals (Sweden)

Bajcsy Peter

2006-01-01

Full Text Available This paper overviews DNA microarray grid alignment and foreground separation approaches. Microarray grid alignment and foreground separation are the basic processing steps of DNA microarray images that affect the quality of gene expression information, and hence impact our confidence in any data-derived biological conclusions. Thus, understanding microarray data processing steps becomes critical for performing optimal microarray data analysis. In the past, the grid alignment and foreground separation steps have not been covered extensively in the survey literature. We present several classifications of existing algorithms, and describe the fundamental principles of these algorithms. Challenges related to automation and reliability of processed image data are outlined at the end of this overview paper.

Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Science.gov (United States)

Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

2016-01-01

Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

OpenAIRE

Jensen, O N; Kulkarni, S; Aldrich, J V; Barofsky, D F

1996-01-01

Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research an...

The application of DNA microarrays in gene expression analysis.

Science.gov (United States)

van Hal, N L; Vorst, O; van Houwelingen, A M; Kok, E J; Peijnenburg, A; Aharoni, A; van Tunen, A J; Keijer, J

2000-03-31

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Rapid Induction of Protective Immunity Against Biothreat Agents Using CPG-Based Oligonucleotides

National Research Council Canada - National Science Library

Klinman, Dennis

2003-01-01

This research project examines the ability of synthetic oligonucleotides (ODN) containing immunostimulatory "CpG motifs' to trigger the innate immune system, thereby improving the host's ability to survive infection by biowarfare agents...

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

DEFF Research Database (Denmark)

Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj

2010-01-01

-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

International Nuclear Information System (INIS)

Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

2002-01-01

Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

Implementing an online tool for genome-wide validation of survival-associated biomarkers in ovarian-cancer using microarray data from 1287 patients

DEFF Research Database (Denmark)

Győrffy, Balázs; Lánczky, András; Szállási, Zoltán

2012-01-01

was set up using gene expression data and survival information of 1287 ovarian cancer patients downloaded from Gene Expression Omnibus and The Cancer Genome Atlas (Affymetrix HG-U133A, HG-U133A 2.0, and HG-U133 Plus 2.0 microarrays). After quality control and normalization, only probes present on all......). A Kaplan–Meier survival plot was generated and significance was computed. The tool can be accessed online at www.kmplot.com/ovar. We used this integrative data analysis tool to validate the prognostic power of 37 biomarkers identified in the literature. Of these, CA125 (MUC16; P=3.7x10–5, hazard ratio (HR...... biomarker validation platform that mines all available microarray data to assess the prognostic power of 22 277 genes in 1287 ovarian cancer patients. We specifically used this tool to evaluate the effect of 37 previously published biomarkers on ovarian cancer prognosis....

Novel approaches to study low-energy electron-induced damage to DNA oligonucleotides

International Nuclear Information System (INIS)

Rackwitz, Jenny; Bald, Ilko; Ranković, Miloš Lj; Milosavljević, Aleksandar R

2015-01-01

The novel approach of DNA origami structures as templates for precise quantification of various well- defined oligonucleotides provides the opportunity to determine the sensitivity of complex DNA sequences towards low-energy electrons. (paper)

Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

International Nuclear Information System (INIS)

Muller, E.

2002-01-01

Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

Bioinformatics on the Cloud Computing Platform Azure

Science.gov (United States)

Shanahan, Hugh P.; Owen, Anne M.; Harrison, Andrew P.

2014-01-01

We discuss the applicability of the Microsoft cloud computing platform, Azure, for bioinformatics. We focus on the usability of the resource rather than its performance. We provide an example of how R can be used on Azure to analyse a large amount of microarray expression data deposited at the public database ArrayExpress. We provide a walk through to demonstrate explicitly how Azure can be used to perform these analyses in Appendix S1 and we offer a comparison with a local computation. We note that the use of the Platform as a Service (PaaS) offering of Azure can represent a steep learning curve for bioinformatics developers who will usually have a Linux and scripting language background. On the other hand, the presence of an additional set of libraries makes it easier to deploy software in a parallel (scalable) fashion and explicitly manage such a production run with only a few hundred lines of code, most of which can be incorporated from a template. We propose that this environment is best suited for running stable bioinformatics software by users not involved with its development. PMID:25050811

Microarray-based large scale detection of single feature ...

Indian Academy of Sciences (India)

2015-12-08

Dec 8, 2015 ... mental stages was used to identify single feature polymorphisms (SFPs). ... on a high-density oligonucleotide expression array in which. ∗ ..... The sign (+/−) with SFPs indicates direction of polymorphism. In the. (−) sign (i.e. ...

Automated synthesis of an {sup 18}F-labelled pyridine-based alkylating agent for high yield oligonucleotide conjugation

Energy Technology Data Exchange (ETDEWEB)

Guggenberg, Elisabeth von; Sader, Jayden A.; Wilson, John S.; Shahhosseini, Soraya; Koslowsky, Ingrid; Wuest, Frank [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada); Mercer, John R. [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada)], E-mail: johnmerc@cancerboard.ab.ca

2009-09-15

Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[{sup 18}F]fluoropyridin-3-yloxy)propyl]acetamide ([{sup 18}F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/{mu}moL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

DEFF Research Database (Denmark)

Astakhova, Irina; Kumar, Santhosh T.; Wengel, Jesper

2011-01-01

efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards...... incorporations of monomers T* was quenched (quantum yield Phi(F) = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Phi(F) = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues...... sequences containing monomers T* at 5'- and 3'-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose...

A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Nookaew, Intawat; Papini, Marta; Pornputtapong, Natapol

2012-01-01

RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated with the I......RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated...... gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays...

Microarrays in brain research: the good, the bad and the ugly.

Science.gov (United States)

Mirnics, K

2001-06-01

Making sense of microarray data is a complex process, in which the interpretation of findings will depend on the overall experimental design and judgement of the investigator performing the analysis. As a result, differences in tissue harvesting, microarray types, sample labelling and data analysis procedures make post hoc sharing of microarray data a great challenge. To ensure rapid and meaningful data exchange, we need to create some order out of the existing chaos. In these ground-breaking microarray standardization and data sharing efforts, NIH agencies should take a leading role

Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Directory of Open Access Journals (Sweden)

Seyedeh Maryam Banihashemian

Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

A comparative analysis of existing oligonucleotides selection ...

African Journals Online (AJOL)

SERVER

2007-07-04

Jul 4, 2007 ... Available online at http://www.academicjournals.org/AJB ... Department of Computer and Information Sciences, Covenant University P. M. B 1023, Ota, Nigeria. ... Key words: System biology, microarray technology, oligo, genome, coding sequence, ... ficity) and efficiency (computer speed and memory requi-.

Advanced Data Mining of Leukemia Cells Micro-Arrays

Directory of Open Access Journals (Sweden)

Richard S. Segall

2009-12-01

Full Text Available This paper provides continuation and extensions of previous research by Segall and Pierce (2009a that discussed data mining for micro-array databases of Leukemia cells for primarily self-organized maps (SOM. As Segall and Pierce (2009a and Segall and Pierce (2009b the results of applying data mining are shown and discussed for the data categories of microarray databases of HL60, Jurkat, NB4 and U937 Leukemia cells that are also described in this article. First, a background section is provided on the work of others pertaining to the applications of data mining to micro-array databases of Leukemia cells and micro-array databases in general. As noted in predecessor article by Segall and Pierce (2009a, micro-array databases are one of the most popular functional genomics tools in use today. This research in this paper is intended to use advanced data mining technologies for better interpretations and knowledge discovery as generated by the patterns of gene expressions of HL60, Jurkat, NB4 and U937 Leukemia cells. The advanced data mining performed entailed using other data mining tools such as cubic clustering criterion, variable importance rankings, decision trees, and more detailed examinations of data mining statistics and study of other self-organized maps (SOM clustering regions of workspace as generated by SAS Enterprise Miner version 4. Conclusions and future directions of the research are also presented.

Significance analysis of lexical bias in microarray data

Directory of Open Access Journals (Sweden)

Falkow Stanley

2003-04-01

Full Text Available Abstract Background Genes that are determined to be significantly differentially regulated in microarray analyses often appear to have functional commonalities, such as being components of the same biochemical pathway. This results in certain words being under- or overrepresented in the list of genes. Distinguishing between biologically meaningful trends and artifacts of annotation and analysis procedures is of the utmost importance, as only true biological trends are of interest for further experimentation. A number of sophisticated methods for identification of significant lexical trends are currently available, but these methods are generally too cumbersome for practical use by most microarray users. Results We have developed a tool, LACK, for calculating the statistical significance of apparent lexical bias in microarray datasets. The frequency of a user-specified list of search terms in a list of genes which are differentially regulated is assessed for statistical significance by comparison to randomly generated datasets. The simplicity of the input files and user interface targets the average microarray user who wishes to have a statistical measure of apparent lexical trends in analyzed datasets without the need for bioinformatics skills. The software is available as Perl source or a Windows executable. Conclusion We have used LACK in our laboratory to generate biological hypotheses based on our microarray data. We demonstrate the program's utility using an example in which we confirm significant upregulation of SPI-2 pathogenicity island of Salmonella enterica serovar Typhimurium by the cation chelator dipyridyl.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

DEFF Research Database (Denmark)

Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

2014-01-01

, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we...

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

Directory of Open Access Journals (Sweden)

Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Facilitating functional annotation of chicken microarray data

Directory of Open Access Journals (Sweden)

Gresham Cathy R

2009-10-01

Full Text Available Abstract Background Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO. However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information. Results We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (AGOM tool to help researchers to quickly retrieve corresponding functional information for their dataset. Conclusion Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using AGOM tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

DEFF Research Database (Denmark)

Pedersen, Henriette Lodberg; Fangel, Jonatan Ulrik; McCleary, Barry

2012-01-01

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less establish...

The application of DNA microarrays in gene expression analysis

NARCIS (Netherlands)

Hal, van N.L.W.; Vorst, O.; Houwelingen, van A.M.M.L.; Kok, E.J.; Peijnenburg, A.A.C.M.; Aharoni, A.; Tunen, van A.J.; Keijer, J.

2000-01-01

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed.

Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

Science.gov (United States)

Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

2005-10-06

We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

KAUST Repository

Wong, Ka-Chun; Peng, Chengbin; Li, Yue

2016-01-01

Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

KAUST Repository

Wong, Ka-Chun

2016-02-02

Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

Science.gov (United States)

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

Microarray of DNA probes on carboxylate functional beads surface

Institute of Scientific and Technical Information of China (English)

黄承志; 李原芳; 黄新华; 范美坤

2000-01-01

The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

Microarray of DNA probes on carboxylate functional beads surface

Institute of Scientific and Technical Information of China (English)

无

2000-01-01

The microarray of DNA probes with 5′-NH2 and 5′-Tex/3′-NH2 modified terminus on 10 m m carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is characterized in the present paper. It was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentration of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca, a drinking water source in the north-eastern part of Turkey.

Science.gov (United States)

Akçaalan, Reyhan; Albay, Meric; Koker, Latife; Baudart, Julia; Guillebault, Delphine; Fischer, Sabine; Weigel, Wilfried; Medlin, Linda K

2017-12-22

Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project μAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.

A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides.

Science.gov (United States)

Su, Xiaoye; Liang, Ruiting; Stolee, Jessica A

2018-06-05

Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides. Copyright © 2018 Elsevier B.V. All rights reserved.

Microarrays for Universal Detection and Identification of Phytoplasmas

DEFF Research Database (Denmark)

Nicolaisen, Mogens; Nyskjold, Henriette; Bertaccini, Assunta

2013-01-01

Detection and identification of phytoplasmas is a laborious process often involving nested PCR followed by restriction enzyme analysis and fine-resolution gel electrophoresis. To improve throughput, other methods are needed. Microarray technology offers a generic assay that can potentially detect...... and differentiate all types of phytoplasmas in one assay. The present protocol describes a microarray-based method for identification of phytoplasmas to 16Sr group level....

Parallel scan hyperspectral fluorescence imaging system and biomedical application for microarrays

International Nuclear Information System (INIS)

Liu Zhiyi; Ma Suihua; Liu Le; Guo Jihua; He Yonghong; Ji Yanhong

2011-01-01

Microarray research offers great potential for analysis of gene expression profile and leads to greatly improved experimental throughput. A number of instruments have been reported for microarray detection, such as chemiluminescence, surface plasmon resonance, and fluorescence markers. Fluorescence imaging is popular for the readout of microarrays. In this paper we develop a quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging system for microarray research. Hyperspectral imaging records the entire emission spectrum for every voxel within the imaged area in contrast to recording only fluorescence intensities of filter-based scanners. Coupled with data analysis, the recorded spectral information allows for quantitative identification of the contributions of multiple, spectrally overlapping fluorescent dyes and elimination of unwanted artifacts. The mechanism of quasi-confocal imaging provides a high signal-to-noise ratio, and parallel scan makes this approach a high throughput technique for microarray analysis. This system is improved with a specifically designed spectrometer which can offer a spectral resolution of 0.2 nm, and operates with spatial resolutions ranging from 2 to 30 μm . Finally, the application of the system is demonstrated by reading out microarrays for identification of bacteria.

An Introduction to MAMA (Meta-Analysis of MicroArray data) System.

Science.gov (United States)

Zhang, Zhe; Fenstermacher, David

2005-01-01

Analyzing microarray data across multiple experiments has been proven advantageous. To support this kind of analysis, we are developing a software system called MAMA (Meta-Analysis of MicroArray data). MAMA utilizes a client-server architecture with a relational database on the server-side for the storage of microarray datasets collected from various resources. The client-side is an application running on the end user's computer that allows the user to manipulate microarray data and analytical results locally. MAMA implementation will integrate several analytical methods, including meta-analysis within an open-source framework offering other developers the flexibility to plug in additional statistical algorithms.

DNA Chip

Indian Academy of Sciences (India)

volumes of data requires techniques which necessitate miniatur- ization and ... of oligonucleotides in search of an elusive gene of interest. The ... multiplexing, automation, and obviously, miniaturization. So, ... with the help of a microarray.

Effective intracellular delivery of oligonucleotides in order to make sense of antisense

NARCIS (Netherlands)

Shi, FX; Hoekstra, D

2004-01-01

For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

CROPPER: a metagene creator resource for cross-platform and cross-species compendium studies.

Science.gov (United States)

Paananen, Jussi; Storvik, Markus; Wong, Garry

2006-09-22

Current genomic research methods provide researchers with enormous amounts of data. Combining data from different high-throughput research technologies commonly available in biological databases can lead to novel findings and increase research efficiency. However, combining data from different heterogeneous sources is often a very arduous task. These sources can be different microarray technology platforms, genomic databases, or experiments performed on various species. Our aim was to develop a software program that could facilitate the combining of data from heterogeneous sources, and thus allow researchers to perform genomic cross-platform/cross-species studies and to use existing experimental data for compendium studies. We have developed a web-based software resource, called CROPPER that uses the latest genomic information concerning different data identifiers and orthologous genes from the Ensembl database. CROPPER can be used to combine genomic data from different heterogeneous sources, allowing researchers to perform cross-platform/cross-species compendium studies without the need for complex computational tools or the requirement of setting up one's own in-house database. We also present an example of a simple cross-platform/cross-species compendium study based on publicly available Parkinson's disease data derived from different sources. CROPPER is a user-friendly and freely available web-based software resource that can be successfully used for cross-species/cross-platform compendium studies.

Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

Science.gov (United States)

Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

2016-01-26

Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application.

Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

Science.gov (United States)

Shahmuradyan, Anna; Krull, Ulrich J

2016-03-15

Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

The use of oligonucleotide probes for meningococcal serotype characterization

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SACCHI Claudio Tavares

1998-01-01

Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

cn.FARMS: a latent variable model to detect copy number variations in microarray data with a low false discovery rate.

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Clevert, Djork-Arné; Mitterecker, Andreas; Mayr, Andreas; Klambauer, Günter; Tuefferd, Marianne; De Bondt, An; Talloen, Willem; Göhlmann, Hinrich; Hochreiter, Sepp

2011-07-01

Cost-effective oligonucleotide genotyping arrays like the Affymetrix SNP 6.0 are still the predominant technique to measure DNA copy number variations (CNVs). However, CNV detection methods for microarrays overestimate both the number and the size of CNV regions and, consequently, suffer from a high false discovery rate (FDR). A high FDR means that many CNVs are wrongly detected and therefore not associated with a disease in a clinical study, though correction for multiple testing takes them into account and thereby decreases the study's discovery power. For controlling the FDR, we propose a probabilistic latent variable model, 'cn.FARMS', which is optimized by a Bayesian maximum a posteriori approach. cn.FARMS controls the FDR through the information gain of the posterior over the prior. The prior represents the null hypothesis of copy number 2 for all samples from which the posterior can only deviate by strong and consistent signals in the data. On HapMap data, cn.FARMS clearly outperformed the two most prevalent methods with respect to sensitivity and FDR. The software cn.FARMS is publicly available as a R package at http://www.bioinf.jku.at/software/cnfarms/cnfarms.html.

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

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Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

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John Cooper

2010-03-01

Full Text Available The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs.An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE

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Ile Kristina E

2003-07-01

Full Text Available Abstract Background The ADGE technique is a method designed to magnify the ratios of gene expression before detection. It improves the detection sensitivity to small change of gene expression and requires small amount of starting material. However, the throughput of ADGE is low. We integrated ADGE with DNA microarray (ADGE microarray and compared it with regular microarray. Results When ADGE was integrated with DNA microarray, a quantitative relationship of a power function between detected and input ratios was found. Because of ratio magnification, ADGE microarray was better able to detect small changes in gene expression in a drug resistant model cell line system. The PCR amplification of templates and efficient labeling reduced the requirement of starting material to as little as 125 ng of total RNA for one slide hybridization and enhanced the signal intensity. Integration of ratio magnification, template amplification and efficient labeling in ADGE microarray reduced artifacts in microarray data and improved detection fidelity. The results of ADGE microarray were less variable and more reproducible than those of regular microarray. A gene expression profile generated with ADGE microarray characterized the drug resistant phenotype, particularly with reference to glutathione, proliferation and kinase pathways. Conclusion ADGE microarray magnified the ratios of differential gene expression in a power function, improved the detection sensitivity and fidelity and reduced the requirement for starting material while maintaining high throughput. ADGE microarray generated a more informative expression pattern than regular microarray.

Hetero-oligonucleotide Nanoscale Tiles Capable of Two-Dimensional Lattice Formation as Testbeds for a Rapid, Affordable Purification Methodology

Science.gov (United States)

2013-01-01

devices; however, DNA is not the only polymer that can take advantage of the specicity of the Watson – Crick base-pair to achieve these goals. Central to...14. ABSTRACT 16. SECURITY CLASSIFICATION OF: New nanoscale hetero-oligonucleotide tiles are assembled from DNA , RNA and morpholino oligos and...purified using size exclusion filtration. Homo-oligonucleotide tiles assembled from RP-cartridge processed DNA oligos are purified by nondenaturing gel

Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

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