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Sample records for adapting dna microarray

  1. DNA Microarray Technique

    Thakare SP

    2012-11-01

    Full Text Available DNA Microarray is the emerging technique in Biotechnology. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. It also includes screening and diagnostic applications. The DNA microarray hybridization applications include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs, and short tandem repeats (STRs. In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes.

  2. Compressive Sensing DNA Microarrays

    Sheikh Mona A

    2009-01-01

    Full Text Available Compressive sensing microarrays (CSMs are DNA-based sensors that operate using group testing and compressive sensing (CS principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed.

  3. DNA Microarray-Based Diagnostics.

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  4. DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

    Hovey, Raymond; Lentes, Sabine; Ehrenreich, Armin; Salmon, Kirsty; Saba, Karla; Gottschalk, Gerhard; Gunsalus, Robert P; Deppenmeier, Uwe

    2005-05-01

    Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes. PMID:15902489

  5. Integrating data from heterogeneous DNA microarray platforms

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set...

  6. Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

    McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

    2015-07-01

    The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland. PMID:25631743

  7. Development and Validation of Corynebacterium DNA Microarrays

    Loos, Andrea; Glanemann, Christoph; Willis, Laura B.; O'Brien, Xian M; Lessard, Philip A.; Gerstmeir, Robert; Guillouet, Stéphane; Sinskey, Anthony J.

    2001-01-01

    We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To es...

  8. Identifying Fishes through DNA Barcodes and Microarrays.

    Marc Kochzius

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  9. Gene Expression Analysis Using Agilent DNA Microarrays

    Stangegaard, Michael

    2009-01-01

    Hybridization of labeled cDNA to microarrays is an intuitively simple and a vastly underestimated process. If it is not performed, optimized, and standardized with the same attention to detail as e.g., RNA amplification, information may be overlooked or even lost. Careful balancing of the amount of...... labeled cDNA added to each slide reduces dye-bias and slide to slide variation. Efficient mixing of the hybridization solution throughout the hybridization reaction increases signals several fold. The amount of near perfect target-probe hybrids may be reduced by efficient stringency washes of the...

  10. Integrating data from heterogeneous DNA microarray platforms.

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

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

    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

  12. DNA Microarray Assessment of Putative Borrelia burgdorferi Lipoprotein Genes

    Liang, Fang Ting; Nelson, F. Kenneth; Fikrig, Erol

    2002-01-01

    A DNA microarray containing fragments of 137 Borrelia burgdorferi B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from B. burgdorferi sensu stricto B31, 297, and N40; Borrelia garinii IP90; and Borrelia afzelii P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes enco...

  13. Innovative DNA microarray design for bacterial flora composition evaluation

    Huyghe, Antoine

    2009-01-01

    During the past decade, the advent of new molecular techniques has led to enormous progress in biology, notably with the development of DNA microarray technology. This technology allows monitoring simultaneously the expression of thousands of genes from a given organism. DNA microarrays have been used in a variety of applications, including the characterization of bacteria in biological samples. In this thesis, two distinct DNA microarray approaches for the characterization of bacterial flora...

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

    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 b...

  15. A comparative analysis of DNA barcode microarray feature size

    Ammar, Ron; SMITH, ANDREW M.; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

    2009-01-01

    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 platfor...

  16. Probe Selection for DNA Microarrays using OligoWiz

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

    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...... 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....

  17. Uses of Dendrimers for DNA Microarrays

    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.

  18. A comparative analysis of DNA barcode microarray feature size

    Smith Andrew M; Ammar Ron; Heisler Lawrence E; Giaever Guri; Nislow Corey

    2009-01-01

    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 microarra...

  19. DNA Microarray Data Analysis: A Novel Biclustering Algorithm Approach

    Tewfik Ahmed H

    2006-01-01

    Full Text Available Biclustering algorithms refer to a distinct class of clustering algorithms that perform simultaneous row-column clustering. Biclustering problems arise in DNA microarray data analysis, collaborative filtering, market research, information retrieval, text mining, electoral trends, exchange analysis, and so forth. When dealing with DNA microarray experimental data for example, the goal of biclustering algorithms is to find submatrices, that is, subgroups of genes and subgroups of conditions, where the genes exhibit highly correlated activities for every condition. In this study, we develop novel biclustering algorithms using basic linear algebra and arithmetic tools. The proposed biclustering algorithms can be used to search for all biclusters with constant values, biclusters with constant values on rows, biclusters with constant values on columns, and biclusters with coherent values from a set of data in a timely manner and without solving any optimization problem. We also show how one of the proposed biclustering algorithms can be adapted to identify biclusters with coherent evolution. The algorithms developed in this study discover all valid biclusters of each type, while almost all previous biclustering approaches will miss some.

  20. DNA Microarray Data Analysis: A Novel Biclustering Algorithm Approach

    Tchagang, Alain B.; Tewfik, Ahmed H.

    2006-12-01

    Biclustering algorithms refer to a distinct class of clustering algorithms that perform simultaneous row-column clustering. Biclustering problems arise in DNA microarray data analysis, collaborative filtering, market research, information retrieval, text mining, electoral trends, exchange analysis, and so forth. When dealing with DNA microarray experimental data for example, the goal of biclustering algorithms is to find submatrices, that is, subgroups of genes and subgroups of conditions, where the genes exhibit highly correlated activities for every condition. In this study, we develop novel biclustering algorithms using basic linear algebra and arithmetic tools. The proposed biclustering algorithms can be used to search for all biclusters with constant values, biclusters with constant values on rows, biclusters with constant values on columns, and biclusters with coherent values from a set of data in a timely manner and without solving any optimization problem. We also show how one of the proposed biclustering algorithms can be adapted to identify biclusters with coherent evolution. The algorithms developed in this study discover all valid biclusters of each type, while almost all previous biclustering approaches will miss some.

  1. cDNA microarray screening in food safety

    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

  2. DNA Microarray Data Analysis: A Novel Biclustering Algorithm Approach

    Tewfik Ahmed H; Tchagang Alain B

    2006-01-01

    Biclustering algorithms refer to a distinct class of clustering algorithms that perform simultaneous row-column clustering. Biclustering problems arise in DNA microarray data analysis, collaborative filtering, market research, information retrieval, text mining, electoral trends, exchange analysis, and so forth. When dealing with DNA microarray experimental data for example, the goal of biclustering algorithms is to find submatrices, that is, subgroups of genes and subgroups of conditions, w...

  3. Microarrays/DNA Chips for the Detection of Waterborne Pathogens.

    Vale, Filipa F

    2016-01-01

    DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis. PMID:27460375

  4. Background Adjustment for DNA Microarrays Using a Database of Microarray Experiments

    Sui, Yunxia; Zhao, Xiaoyue; Speed, Terence P.; Wu, Zhijin

    2009-01-01

    DNA microarrays have become an indispensable technique in biomedical research. The raw measurements from microarrays undergo a number of preprocessing steps before the data are converted to the genomic level for further analysis. Background adjustment is an important step in preprocessing. Estimating background noise has been challenging because background levels vary a lot from probe to probe, yet there are limited observations on each probe. Most current methods have used the empirical Baye...

  5. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions

  6. AFM 4.0: a toolbox for DNA microarray analysis

    Breitkreutz, Bobby-Joe; Jorgensen, Paul; Breitkreutz, Ashton; Tyers, Mike

    2001-01-01

    We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to ...

  7. Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

    Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

    2007-01-01

    We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promisi...

  8. Array-A-Lizer: A serial DNA microarray quality analyzer

    Matthiessen Mads

    2004-02-01

    Full Text Available Abstract Background The proliferate nature of DNA microarray results have made it necessary to implement a uniform and quick quality control of experimental results to ensure the consistency of data across multiple experiments prior to actual data analysis. Results Array-A-Lizer is a small and convenient stand-alone tool providing the necessary initial analysis of hybridization quality of an unlimited number of microarray experiments. The experiments are analyzed for even hybridization across the slide and between fluorescent dyes in two-color experiments in spotted DNA microarrays. Conclusions Array-A-Lizer allows the expedient determination of the quality of multiple DNA microarray experiments allowing for a rapid initial screening of results before progressing to further data analysis. Array-A-Lizer is directed towards speed and ease-of-use allowing both the expert and non-expert microarray researcher to rapidly assess the quality of multiple microarray hybridizations. Array-A-Lizer is available from the Internet as both source code and as a binary installation package.

  9. A meta-data based method for DNA microarray imputation

    Ouyang Ming

    2007-03-01

    Full Text Available Abstract Background DNA microarray experiments are conducted in logical sets, such as time course profiling after a treatment is applied to the samples, or comparisons of the samples under two or more conditions. Due to cost and design constraints of spotted cDNA microarray experiments, each logical set commonly includes only a small number of replicates per condition. Despite the vast improvement of the microarray technology in recent years, missing values are prevalent. Intuitively, imputation of missing values is best done using many replicates within the same logical set. In practice, there are few replicates and thus reliable imputation within logical sets is difficult. However, it is in the case of few replicates that the presence of missing values, and how they are imputed, can have the most profound impact on the outcome of downstream analyses (e.g. significance analysis and clustering. This study explores the feasibility of imputation across logical sets, using the vast amount of publicly available microarray data to improve imputation reliability in the small sample size setting. Results We download all cDNA microarray data of Saccharomyces cerevisiae, Arabidopsis thaliana, and Caenorhabditis elegans from the Stanford Microarray Database. Through cross-validation and simulation, we find that, for all three species, our proposed imputation using data from public databases is far superior to imputation within a logical set, sometimes to an astonishing degree. Furthermore, the imputation root mean square error for significant genes is generally a lot less than that of non-significant ones. Conclusion Since downstream analysis of significant genes, such as clustering and network analysis, can be very sensitive to small perturbations of estimated gene effects, it is highly recommended that researchers apply reliable data imputation prior to further analysis. Our method can also be applied to cDNA microarray experiments from other species

  10. DNA microarray technique for detecting food-borne pathogens

    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.

  11. Development of DNA Microarrays for Metabolic Pathway and Bioprocess Monitoring

    Gregory Stephanopoulos

    2004-07-31

    Transcriptional profiling experiments utilizing DNA microarrays to study the intracellular accumulation of PHB in Synechocystis has proved difficult in large part because strains that show significant differences in PHB which would justify global analysis of gene expression have not been isolated.

  12. DNA microarray analysis of fim mutations in Escherichia coli

    Schembri, Mark; Ussery, David; Workman, Christopher; Hasman, Henrik; Klemm, Per

    2002-01-01

    we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  13. A comparative analysis of DNA barcode microarray feature size

    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.

  14. Granulometric Analysis of Spots in DNA Microarray Images

    Behara Latha; Balasubramanian Venkatesh

    2004-01-01

    As the topological properties of each spot in DNA microarray images may vary from one another, we employed granulometries to understand the shape-size con tent contributed due to a significant intensity value within a spot. Analysis was performed on the microarray image that consisted of 240 spots by using concepts from mathematical morphology. In order to find out indices for each spot and to further classify them, we adopted morphological multiscale openings, which provided microarrays at multiple scales. Successive opened microarrays were subtracted to identify the protrusions that were smaller than the size of structuring element. Spot-wise details, in terms of probability of these observed protrusions,were computed by placing a regularly spaced grid on microarray such that each spot was centered in each grid. Based on the probability of size distribution functions of these protrusions isolated at each level, we estimated the mean size and texture index for each spot. With these characteristics, we classified the spots in a microarray image into bright and dull categories through pattern spectrum and shape-size complexity measures. These segregated spots can be compared with those of hybridization levels.

  15. An Overview of DNA Microarray Grid Alignment and Foreground Separation Approaches

    Bajcsy Peter

    2006-01-01

    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 s...

  16. DNA microarray: parallel detection of potato viruses

    Bystřická, Dagmar; Lenz, Ondřej; Mráz, Ivan; Dědič, P.; Šíp, M.

    2003-01-01

    Roč. 47, - (2003), s. 41-44. ISSN 0001-723X R&D Projects: GA ČR GA522/01/1105 Institutional research plan: CEZ:AV0Z5051902 Keywords : potato viruses, DNA Subject RIV: EE - Microbiology, Virology Impact factor: 0.683, year: 2003

  17. Detection of bacterial pathogens in environmental samples using DNA microarrays.

    Call, Douglas R; Borucki, Monica K; Loge, Frank J

    2003-05-01

    Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes. PMID:12654494

  18. DNA Microarrays in Comparative Genomics and Transcriptomics

    Willenbrock, Hanni

    2007-01-01

    analysis, analysis of chromosomal aberrations and DNA sequence dependent gene expression. First, this thesis contains a description of how the gene expression profiles from children with acute lymphoblastic leukemia may be used to improve the diagnosis of these patients and potentially improve their...... experimental factor such as compound treatment may be obtained. The same characterization could otherwise be time consuming and require an extensive biological knowledge of the investigated biological system. Often, solid tumors are characterized by a multitude of chromosomal aberrations where parts of the...... verify predictions of highly expressed genes. Moreover, the codon bias of microbial genomes was found to constitute an environmental signature. For example, soil bacteria have very similar codon bias....

  19. DNA-Microarray-based Genotyping of Clostridium difficile

    Gawlik, Darius; Slickers, Peter; Engelmann, Ines; Müller, Elke; Lück, Christian; Friedrichs, Anette; Ehricht, Ralf; Monecke, Stefan

    2015-01-01

    Background Clostridium difficile can cause antibiotic-associated diarrhea and a possibility of outbreaks in hospital settings warrants molecular typing. A microarray was designed that included toxin genes (tcdA/B, cdtA/B), genes related to antimicrobial resistance, the slpA gene and additional variable genes. Results DNA of six reference strains and 234 clinical isolates from South-Western and Eastern Germany was subjected to linear amplification and labeling with dUTP-linked biotin. Amplicon...

  20. The Widely Used Diagnostics "DNA Microarray"-A Review

    Kumar, R M

    2009-01-01

    Problem statement: DNA microarray technique is one of the latest advances in the field of molecular biology and medicine. It is a multiplex technique used in combination of bioinformatics and statistical data analysis. Since, 1995, the technique offers the possibility of conducting tens or hundreds of thousands of simultaneous hybridizations. Approach: This increased experimental efficiency permits high throughput and whole genome expression profiling of pathogen...

  1. Gene networks from DNA microarray data: centrality and lethality

    Provero, P.

    2002-01-01

    We construct a gene network based on expression data from DNA microarray experiments, by establishing a link between two genes whenever the Pearson's correlation coefficient between their expression profiles is higher than a certain cutoff. The resulting connectivity distribution is compatible with a power-law decay with exponent ~1, corrected by an exponential cutoff at large connectivity. The biological relevance of such network is demonstrated by showing that there is a strong statistical ...

  2. Combining DNA-microarray data in systemic lupus erythematosus

    Verweij, Cornelis L.; Vosslamber, Saskia

    2011-01-01

    Systemic lupus erythematosus is a systemic, heterogeneous autoimmune disease. Understanding of its molecular complexity is incomplete and there is a need to identify new therapeutic targets and to optimize criteria for its diagnosis, assessment and prognosis. Recently, Arasappan and colleagues have described a new meta-analysis method that enables data analysis across different DNA-microarray datasets to identify genes and processes relevant to systemic lupus erythematosus. Their study provid...

  3. DNA Microarray Technologies: A Novel Approach to Geonomic Research

    Hinman, R.; Thrall, B.; Wong, K,

    2002-01-01

    A cDNA microarray allows biologists to examine the expression of thousands of genes simultaneously. Researchers may analyze the complete transcriptional program of an organism in response to specific physiological or developmental conditions. By design, a cDNA microarray is an experiment with many variables and few controls. One question that inevitably arises when working with a cDNA microarray is data reproducibility. How easy is it to confirm mRNA expression patterns? In this paper, a case study involving the treatment of a murine macrophage RAW 264.7 cell line with tumor necrosis factor alpha (TNF) was used to obtain a rough estimate of data reproducibility. Two trials were examined and a list of genes displaying either a > 2-fold or > 4-fold increase in gene expression was compiled. Variations in signal mean ratios between the two slides were observed. We can assume that erring in reproducibility may be compensated by greater inductive levels of similar genes. Steps taken to obtain results included serum starvation of cells before treatment, tests of mRNA for quality/consistency, and data normalization.

  4. Thermodynamics of competitive surface adsorption on DNA microarrays

    Gene microarrays provide a powerful functional genomics technology which permits the expression profiling of tens of thousands of genes in parallel. The basic idea of their functioning is based on the sequence specificity of probe-target interactions combined with fluorescence detection. In reality, this straightforward principle is opposed by the complexity of the experimental system due to imperfections of chip fabrication and RNA preparation, due to the non-linearity of the probe response and especially due to competitive interactions which are inherently connected with the high throughput character of the method. We theoretically analysed aspects of the hybridization of DNA oligonucleotide probes with a complex multicomponent mixture of RNA fragments, such as the effect of different interactions between nucleotide strands competing with the formation of specific duplexes, electrostatic and entropic blocking, the fragmentation of the RNA, the incomplete synthesis of the probes and 'zipping' effects in the oligonucleotide duplexes. The effective hybridization affinities of microarray probes are considerably smaller than those for bulk hybridization owing to the effects discussed, but they correlate well with the bulk data on a relative scale. In general, the hybridization isotherms of microarray probes are shown to deviate from a Langmuir-type behaviour. Nevertheless isotherms of the Langmuir or Sips type are predicted to provide a relatively simple description of the non-linear, probe-specific concentration dependence of the signal intensity of microarray probes

  5. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    TENG XiaoKun; XIAO HuaSheng

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequenc-ing method was first introduced by the 454 Company in 2003, immediately followed by the establish-ment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  6. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research,in revealing both the structural and functional characteristics of genomes.In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics,systems biology and pharmacogenomics.The next-generation DNA sequencing method was first introduced by the 454 Company in 2003,immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies.Though it has not been long since the first emergence of this technology,with the fast and impressive improvement,the application of this technology has extended to almost all fields of genomics research,as a rival challenging the existing DNA microarray technology.This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  7. Investigating amoebic pathogenesis using Entamoeba histolytica DNA microarrays

    Upinder Singh; Preetam Shah

    2002-11-01

    Entamoeba histolytica, a protozoan parasite, causes diarrhea and liver abscesses resulting in 50 million cases of infection worldwide annually. Elucidation of parasite virulence determinants has recently been investigated using genetic approaches. We have undertaken a genomics approach to identify novel virulence determinants in the parasite. A DNA microarray of E. histolytica is being developed based on sequenced genomic clones from the genome sequencing efforts of The Institute of Genomic Research (TIGR) and the Sanger Center. Hybridization of the slides with samples labelled differentially using fluorescent dyes allows the characterization of transcriptional profiles of genes under the biological conditions tested. Additionally, a genome-wide comparison of E. histolytica and E. dispar can be undertaken. The development of an E. histolytica microarray will be outlined and its uses in identifying novel virulence determinants and characterizing amoebic biology will be discussed.

  8. Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

    Jennifer A. Martin

    2015-01-01

    Full Text Available A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

  9. Development of a Daphnia magna DNA microarray for evaluating the toxicity of environmental chemicals.

    Watanabe, Hajime; Takahashi, Eri; Nakamura, Yuko; Oda, Shigeto; Tatarazako, Norihisa; Iguchi, Taisen

    2007-04-01

    Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism- and population-level responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna. Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide-based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or beta-naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical-specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism. PMID:17447551

  10. DNA microarray analysis of fim mutations in Escherichia coli

    Schembri, Mark; Ussery, David; Workman, Christopher;

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated with...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  11. GenePublisher: automated analysis of DNA microarray data

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.;

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization......, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user....

  12. Subtype Identification of Avian Influenza Virus on DNA Microarray

    WANG Xiu-rong; YU Kang-zhen; DENG Guo-hua; SHI Rui; LIU Li-ling; QIAO Chuan-ling; BAO Hong-mei; KONG Xian-gang; CHEN Hua-lan

    2005-01-01

    We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus (AIV). The strains used in the experiment were A/Goose/Guangdong/1/96 (H5N1), A/African starling/983/79 (H7N1) and A/Turkey/Wiscosin/1/66 (H9N2). The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-1abeled fluorescent cDNAs,which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

  13. An algorithm for automatic evaluation of the spot quality in two-color DNA microarray experiments

    Barillot Emmanuel; Novikov Eugene

    2005-01-01

    Abstract Background Although DNA microarray technologies are very powerful for the simultaneous quantitative characterization of thousands of genes, the quality of the obtained experimental data is often far from ideal. The measured microarrays images represent a regular collection of spots, and the intensity of light at each spot is proportional to the DNA copy number or to the expression level of the gene whose DNA clone is spotted. Spot quality control is an essential part of microarray im...

  14. Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

    Gase Klaus

    2004-09-01

    Full Text Available Abstract Background Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER data produced from microarrays. Results We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV were used to calculate array-based variances (array CV, which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA

  15. Extraction of Spots in DNA Microarrays Using Genetic Algorithm

    A. Sreedevi

    2013-12-01

    Full Text Available DNA microarray technology is an eminent tool for ge nomic studies. Accurate extraction of spots is a crucial issue since biological interpretations depe nd on it. The image analysis starts with the format ion of grid, which is a laborious process requiring human intervention. This paper presents a method for opti mal search of the spots using genetic algorithm without formation of grid. The information of every spot i s extracted by obtaining a pixel belonging to that sp ot. The method developed selects pixels of high int ensity in the image, thereby spot is recognized. The objec tive function, which is implemented, helps in ident ifying the exact pixel. The algorithm is applied to differ ent sizes of sub images and features of the spots a re obtained. It is found that there is a tradeoff betw een accuracy in the number of spots identified and time required for processing the image. Segmentation pro cess is independent of shape, size and location of the spots. Background estimation is one step process as both foreground and complete spot are realized. Coding of the proposed algorithm is developed in MA TLAB-7 and applied to cDNA microarray images. This approach provides reliable results for identif ication of even low intensity spots and elimination of spurious spots.

  16. An Overview of DNA Microarray Grid Alignment and Foreground Separation Approaches

    Bajcsy, Peter

    2006-12-01

    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.

  17. Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE

    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.

  18. Design issues in toxicogenomics using DNA microarray experiment

    The methods of toxicogenomics might be classified into omics study (e.g., genomics, proteomics, and metabolomics) and population study focusing on risk assessment and gene-environment interaction. In omics study, microarray is the most popular approach. Genes falling into several categories (e.g., xenobiotics metabolism, cell cycle control, DNA repair etc.) can be selected up to 20,000 according to a priori hypothesis. The appropriate type of samples and species should be selected in advance. Multiple doses and varied exposure durations are suggested to identify those genes clearly linked to toxic response. Microarray experiments can be affected by numerous nuisance variables including experimental designs, sample extraction, type of scanners, etc. The number of slides might be determined from the magnitude and variance of expression change, false-positive rate, and desired power. Instead, pooling samples is an alternative. Online databases on chemicals with known exposure-disease outcomes and genetic information can aid the interpretation of the normalized results. Gene function can be inferred from microarray data analyzed by bioinformatics methods such as cluster analysis. The population study often adopts hospital-based or nested case-control design. Biases in subject selection and exposure assessment should be minimized, and confounding bias should also be controlled for in stratified or multiple regression analysis. Optimal sample sizes are dependent on the statistical test for gene-to-environment or gene-to-gene interaction. The design issues addressed in this mini-review are crucial in conducting toxicogenomics study. In addition, integrative approach of exposure assessment, epidemiology, and clinical trial is required

  19. Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

    Pourmand, Nader; Caramuta, Stefano; Villablanca, Andrea; Mori, Silvia; Karhanek, Miloslav; Wang, Shan X.; Ronald W Davis

    2007-01-01

    The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics ...

  20. The Widely Used Diagnostics "DNA Microarray"-A Review

    R. M. Kumar

    2009-01-01

    Full Text Available Problem statement: DNA microarray technique is one of the latest advances in the field of molecular biology and medicine. It is a multiplex technique used in combination of bioinformatics and statistical data analysis. Since, 1995, the technique offers the possibility of conducting tens or hundreds of thousands of simultaneous hybridizations. Approach: This increased experimental efficiency permits high throughput and whole genome expression profiling of pathogens and hosts. Results: Microarray technologies are rapidly advancing with numerous applications in gene expression, genotyping, pharmaco-genomics, proteomics and cell biology, in infectious diseases recognizing the causative agent, molecular typing, in studying the interactions between causative agents and host cells, cancer biology, genetics, determining the presence of a pathogen in food samples, to characterize microbial isolates, identifying the presence of virulence factors or microbial resistance genes, to detect microbial mutations associated with resistance to antiretroviral drugs, simultaneously detect and discriminate several viruses, to study the contaminants in cultures, such as mycoplasma, yeasts, fungi, exogenous and endogenous viruses and prions from both animal and human origin so and so forth. Conclusion: This article reviews the applicability of this technique elaborately in some of the important areas of biological sciences.

  1. Direct Detection and Genotyping of KPC Carbapenemases from Urine using a new DNA Microarray Test

    Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate; Bachmann, Till T

    2012-01-01

    Klebsiella pneumoniae carbapenemases (KPC) are considered a serious threat to antibiotic therapy as they confer resistance to carbapenems, which are used to treat Extended-spectrum beta-lactamase (ESBL) producing bacteria. Here, we describe the development and evaluation of a DNA microarray for detection and genotyping of KPC genes (blaKPC) within 5 hours. To test the whole assay procedure (DNA extraction + DNA microarray assay) directly from clinical specimen, we compared two commercial DNA ...

  2. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  3. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  4. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  5. DNA Microarray Wet Lab Simulation Brings Genomics into the High School Curriculum

    Campbell, A. Malcolm; Zanta, Carolyn A.; Heyer, Laurie J.; Kittinger, Ben; Gabric, Kathleen M.; Adler, Leslie

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH indicators, which offer many ideal teaching characteristics. The simulation requires no specialized equipment, is very inexpensive, is very reliable, a...

  6. Experimental genomics: The application of DNA microarrays in cellular and molecular biology studies

    2002-01-01

    The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellular and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quant itative fashion. DNA microarrays can be used to measure levels of gene expressio n for tens of thousands of gene simultaneously and take advantage of all availab le sequence information for experimental design and data interpretation in pursu it of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a catalogue of all the genes and informati on about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in genome and gene function analysis, gene expression studies, biological signal and defense system, cell cyclereg ulation, mechanism of transcriptional regulation, proteomics, and the functional ity of food component.

  7. DNA Microarray Detection of Antimicrobial Resistance Genes in Bacteria Co-Cultured from Swine Feces

    One factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes. To study this, a DNA microarray was recently developed to detect these genes. To maximize the capability of this microarray, probes were designed and added to detect all AR g...

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

    Dufva, Martin; Petersen, Jesper; Poulsen, Lena

    2009-01-01

    building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit...... microarray-based loci selection prior to high-throughput sequencing....

  9. Fabrication of DNA microarrays on polydopamine-modified gold thin films for SPR imaging measurements.

    Wood, Jennifer B; Szyndler, Megan W; Halpern, Aaron R; Cho, Kyunghee; Corn, Robert M

    2013-08-27

    Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultrasensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with non-complementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid, and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing. PMID:23902428

  10. The effect of column purification on cDNA indirect labelling for microarrays

    Kiss John Z

    2007-06-01

    Full Text Available Abstract Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive

  11. Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

    王峰

    2013-01-01

    Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium

  12. Microarray long oligo probe designing for Escherichia coli: an in-silico DNA marker extraction

    Behzadi, Payam; Najafi, Ali; BEHZADI, Elham; Ranjbar, Reza

    2016-01-01

    Introduction Urinary tract infections are predominant diseases which may be caused by different pathogenic microorganisms, particularly Escherichia coli (E.coli). DNA microarray technology is an accurate, rapid, sensitive, and specific diagnostic tool which may lead to definite diagnosis and treatment of several infectious diseases. DNA microarray is a multi-process method in which probe designing plays an important. Therefore, the authors of the present study have tried to design a range of ...

  13. A biclustering algorithm based on a Bicluster Enumeration Tree: application to DNA microarray data

    Ayadi Wassim; Elloumi Mourad; Hao Jin-Kao

    2009-01-01

    Abstract Background In a number of domains, like in DNA microarray data analysis, we need to cluster simultaneously rows (genes) and columns (conditions) of a data matrix to identify groups of rows coherent with groups of columns. This kind of clustering is called biclustering. Biclustering algorithms are extensively used in DNA microarray data analysis. More effective biclustering algorithms are highly desirable and needed. Methods We introduce BiMine, a new enumeration algorithm for biclust...

  14. DNA Microarray as Part of a Genomic-Assisted Breeding Approach

    Vincze, Éva; Bowra, Steve

    2010-01-01

    be major considerations when the technique is applied. We consider the use of cDNA vs. oligonucleotide microarrays, target purification, labelling, hybridisation, image acquisition, minimising random errors, experimental design, biological and technical variability, quality control, normalisation......, statistical and practical significances, fold changes, validation and possible additional regulatory mechanisms in gene expression. The subject of the fourth section is the applications of DNA microarrays to study of global gene expression during grain filling in monocot crops, especially barley. We compare...

  15. An Overview of DNA Microarray Grid Alignment and Foreground Separation Approaches

    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.

  16. Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses

    Deising Holger B

    2011-01-01

    Full Text Available Abstract Background The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples. Results We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024 groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide

  17. Swarm Intelligence Approach Based on Adaptive ELM Classifier with ICGA Selection for Microarray Gene Expression and Cancer Classification

    T. Karthikeyan

    2014-05-01

    Full Text Available The aim of this research study is based on efficient gene selection and classification of microarray data analysis using hybrid machine learning algorithms. The beginning of microarray technology has enabled the researchers to quickly measure the position of thousands of genes expressed in an organic/biological tissue samples in a solitary experiment. One of the important applications of this microarray technology is to classify the tissue samples using their gene expression representation, identify numerous type of cancer. Cancer is a group of diseases in which a set of cells shows uncontrolled growth, instance that interrupts upon and destroys nearby tissues and spreading to other locations in the body via lymph or blood. Cancer has becomes a one of the major important disease in current scenario. DNA microarrays turn out to be an effectual tool utilized in molecular biology and cancer diagnosis. Microarrays can be measured to establish the relative quantity of mRNAs in two or additional organic/biological tissue samples for thousands/several thousands of genes at the same time. As the superiority of this technique become exactly analysis/identifying the suitable assessment of microarray data in various open issues. In the field of medical sciences multi-category cancer classification play a major important role to classify the cancer types according to the gene expression. The need of the cancer classification has been become indispensible, because the numbers of cancer victims are increasing steadily identified by recent years. To perform this proposed a combination of Integer-Coded Genetic Algorithm (ICGA and Artificial Bee Colony algorithm (ABC, coupled with an Adaptive Extreme Learning Machine (AELM, is used for gene selection and cancer classification. ICGA is used with ABC based AELM classifier to chose an optimal set of genes which results in an efficient hybrid algorithm that can handle sparse data and sample imbalance. The

  18. Correlation Statistics for cDNA Microarray Image Analysis

    Nagarajan, Radhakrishnan; Upreti, Meenakshi

    2005-01-01

    In this report, correlation of the pixels comprising a microarray spot is investigated. Subsequently, correlation statistics namely: Pearson correlation and Spearman rank correlation are used to segment the foreground and background intensity of microarray spots. The performance of correlation-based segmentation is compared to clustering-based (PAM, k-means) and seeded-region growing techniques (SPOT). It is shown that correlation-based segmentation is useful in flagging poorly hybridized spo...

  19. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli. PMID:15304751

  20. Splicing-Sensitive DNA-Microarrays: Peculiarities and Applicationin Biomedical Research (Review

    D.I. Knyazev

    2015-12-01

    Full Text Available Alternative splicing (АS provides a variety of protein and mature mRNA isoforms encoded by a single gene, and is the essential component of cell and tissue differentiation and functioning. DNA-microarrays are highly productive transcriptome research technique both at the level of total gene expression assessment and alternatively spliced mRNA isoforms exploration. The study of AS patterns requires thorough probe design to achieve appropriate accuracy of the analysis. There are two types of splicing-sensitive DNA-microarrays. The first type contain probes targeted to internal exonic sequences (exon bodies; the second type contain probes targeted to exon bodies and exon–exon and exon–intron junctions. So, the first section focused on probe sequence design, general features of splicing-sensitive DNA-microarrays and their main advantages and limitations. The results of AS research obtained using DNA-microarrays have been reviewed in special section. In particular, DNA-microarrays were used to reveal a number pre-mRNA processing and splicing mechanisms, to investigate AS patterns associated with cancer, cell and tissue differentiation. Splicing machinery regulation was demonstrated to be an essential step during carcinogenesis and differentiation. The examples of application of splicing-sensitive DNA-microarrays for diagnostic markers discovering and pathology mechanism elucidation were also reviewed. Investigations of AS role in pluripotency, stem cell commitment, immune and infected cells functioning during immune response are the promising future directions. Splicing-sensitive DNA-microarrays are relatively inexpensive but powerful research tool that give reason to suppose their introduction in clinical practice within the next few years.

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

    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.

  2. Gene expression analysis of strawberry achene and receptacle maturation using DNA microarrays

    Aharoni, A.; O'Connell, A.P.

    2002-01-01

    Large-scale, single pass sequencing and parallel gene expression analysis using DNA microarrays were employed for the comprehensive investigation of ripening in strawberry fruit. A total of 1701 cDNA clones (comprising 1100 strawberry ESTs and 601 unsequenced cDNAs) obtained from a strawberry (Fraga

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

    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. PMID:26922047

  4. DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells

    Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes

  5. Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

    Porro Danilo; Maurer Michael; Valli Minoska; Gasser Brigitte; Branduardi Paola; Sauer Michael; Mattanovich Diethard

    2004-01-01

    Abstract Background Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. Results We could show th...

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

    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

  7. Iterative normalization of cDNA microarray data.

    Wang, Yue; Lu, Jianping; Lee, Richard; Gu, Zhiping; Clarke, Robert

    2002-03-01

    This paper describes a new approach to normalizing microarray expression data. The novel feature is to unify the tasks of estimating normalization coefficients and identifying control gene set. Unification is realized by constructing a window function over the scatter plot defining the subset of constantly expressed genes and by affecting optimization using an iterative procedure. The structure of window function gates contributions to the control gene set used to estimate normalization coefficients. This window measures the consistency of the matched neighborhoods in the scatter plot and provides a means of rejecting control gene outliers. The recovery of normalizational regression and control gene selection are interleaved and are realized by applying coupled operations to the mean square error function. In this way, the two processes bootstrap one another. We evaluate the technique on real microarray data from breast cancer cell lines and complement the experiment with a data cluster visualization study. PMID:11936594

  8. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates

    Nitschke, Heike; Slickers, Peter; Müller, Elke; Ehricht, Ralf; Monecke, Stefan

    2014-01-01

    Streptococcus agalactiae frequently colonizes the urogenital tract, and it is a major cause of bacterial septicemia, meningitis, and pneumonia in newborns. For typing purposes, a microarray targeting group B streptococcus (GBS) virulence-associated markers and resistance genes was designed and validated with reference strains, as well as clinical and veterinary isolates. Selected isolates were also subjected to multilocus sequence typing. It was observed that putative typing markers, such as ...

  9. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.

    Albrecht, Valérie; Chevallier, Anne; Magnone, Virginie; Barbry, Pascal; Vandenbos, Fanny; Bongain, André; Lefebvre, Jean-Claude; Giordanengo, Valérie

    2006-11-01

    Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities. PMID:16879879

  10. DNA microarray for tracing Salmonella in the feed chain.

    Koyuncu, Sevinc; Andersson, Gunnar; Vos, Pieter; Häggblom, Per

    2011-03-01

    In the present study we investigated if the microarray platforms Premi®Test Salmonella (PTS) and Salmonella array (SA) could be applied for the identification and typing of Salmonella in artificially contaminated animal feed materials. The results were compared to the culture-based MSRV method and serotyping according to Kauffman-White. The SA platform showed a specificity of 100% for the identification of Salmonella compared to 93% with the PTS platform and a sensitivity of 99% or 100%, respectively. Among all identified Salmonella serotypes, 56% with the SA platform and 81% with the PTS platform were correctly identified. The difference in probe signal intensity for each probe was higher between duplicates analyzed with the SA platform than with the PTS platform. Attempts to use the microarray platforms from BPW resulted in many false negative samples and incorrect typing results. The microarray platforms tested were simple to use and might have a potential in tracing studies for Salmonella in the feed chain particularly when rapid information about serotypes are important. PMID:20688409

  11. A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

    Mancia, Annalaura; Lundqvist, Mats L; Romano, Tracy A; Peden-Adams, Margie M; Fair, Patricia A; Kindy, Mark S; Ellis, Blake C; Gattoni-Celli, Sebastiano; McKillen, David J; Trent, Harold F; Chen, Yian Ann; Almeida, Jonas S; Gross, Paul S; Chapman, Robert W; Warr, Gregory W

    2007-01-01

    A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues. PMID:17084893

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

    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.

  13. Microarray long oligo probe designing for Escherichia coli: an in-silico DNA marker extraction

    Behzadi, Payam; Najafi, Ali; Behzadi, Elham

    2016-01-01

    Introduction Urinary tract infections are predominant diseases which may be caused by different pathogenic microorganisms, particularly Escherichia coli (E.coli). DNA microarray technology is an accurate, rapid, sensitive, and specific diagnostic tool which may lead to definite diagnosis and treatment of several infectious diseases. DNA microarray is a multi-process method in which probe designing plays an important. Therefore, the authors of the present study have tried to design a range of effective and proper long oligo microarray probes for detection and identification of different strains of pathogenic E.coli and in particular, uropathogenic E.coli (UPEC). Material and methods E.coli O26 H11 11368 uid41021 was selected as the standard strain for probe designing. This strain encompasses the largest nucleotide sequence and the most number of genes among other pathogenic strains of E.coli. For performing this in silico survey, NCBI database, GReview Server, PanSeq Server, Oligoanalyzer tool, and AlleleID 7.7 were used to design accurate, appropriate, effective, and flexible long oligo microarray probes. Moreover, the genome of E.coli and its closely related microorganisms were compared. Results In this study, 15 long oligo microarray probes were designed for detecting and identifying different strains of E.coli such as UPEC. These probes possessed the best physico-chemical characteristics. The functional and structural properties of the designed probes were recognized by practical tools and softwares. Conclusions The use of reliable advanced technologies and methodologies for probe designing guarentees the high quality of microarray probes and makes DNA microarray technology more flexible and an effective diagnostic technique.

  14. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  15. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  16. Use of the cDNA microarray technology in the safety assessment of GM food plants

    Kok, E.J.; Kleter, G.A.; Dijk, van J.P.

    2003-01-01

    This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates the

  17. A biclustering algorithm based on a Bicluster Enumeration Tree: application to DNA microarray data

    Ayadi Wassim

    2009-12-01

    Full Text Available Abstract Background In a number of domains, like in DNA microarray data analysis, we need to cluster simultaneously rows (genes and columns (conditions of a data matrix to identify groups of rows coherent with groups of columns. This kind of clustering is called biclustering. Biclustering algorithms are extensively used in DNA microarray data analysis. More effective biclustering algorithms are highly desirable and needed. Methods We introduce BiMine, a new enumeration algorithm for biclustering of DNA microarray data. The proposed algorithm is based on three original features. First, BiMine relies on a new evaluation function called Average Spearman's rho (ASR. Second, BiMine uses a new tree structure, called Bicluster Enumeration Tree (BET, to represent the different biclusters discovered during the enumeration process. Third, to avoid the combinatorial explosion of the search tree, BiMine introduces a parametric rule that allows the enumeration process to cut tree branches that cannot lead to good biclusters. Results The performance of the proposed algorithm is assessed using both synthetic and real DNA microarray data. The experimental results show that BiMine competes well with several other biclustering methods. Moreover, we test the biological significance using a gene annotation web-tool to show that our proposed method is able to produce biologically relevant biclusters. The software is available upon request from the authors to academic users.

  18. Use of Low-Density DNA Microarrays and Photopolymerization for Genotyping Foodborne-Associated Noroviruses

    Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, the use of low density DNA microarrays in conjunct...

  19. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

    King-Jung Lin

    2012-02-01

    Full Text Available We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP, resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

  20. Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  1. Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays

    Lin, Baochuan; Wang, Zheng; Vora, Gary J.; Jennifer A. Thornton; Schnur, Joel M.; Thach, Dzung C.; Blaney, Kate M.; Ligler, Adam G.; Malanoski, Anthony P.; Santiago, Jose; Walter, Elizabeth A.; Agan, Brian K.; Metzgar, David; Seto, Donald; Daum, Luke T.

    2006-01-01

    The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation st...

  2. QPSO-based adaptive DNA computing algorithm.

    Karakose, Mehmet; Cigdem, Ugur

    2013-01-01

    DNA (deoxyribonucleic acid) computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO). Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1) parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2) adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3) numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm. PMID:23935409

  3. QPSO-Based Adaptive DNA Computing Algorithm

    Mehmet Karakose

    2013-01-01

    Full Text Available DNA (deoxyribonucleic acid computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO. Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1 parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2 adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3 numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm.

  4. The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

    Lemkin, P F; Thornwall, G C; Walton, K D; Hennighausen, L

    2000-11-15

    The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins

  5. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  6. DNA microarray analysis of genes differentially expressed in adipocyte differentiation

    Chunyan Yin; Yanfeng Xiao; Wei Zhang; Erdi Xu; Weihua Liu; Xiaoqing Yi; Ming Chang

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a ≥ 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RT-PCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  7. Xylella fastidiosa gene expression analysis by DNA microarrays

    Regiane F. Travensolo

    2009-01-01

    Full Text Available Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE. All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others. The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.

  8. Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

    Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate; Bachmann, Till T

    2012-01-01

    Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared tw...

  9. Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

    Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate; Bachmann, Till T

    2012-01-01

    Klebsiella pneumoniae carbapenemases (KPC) are considered a serious threat to antibiotic therapy as they confer resistance to carbapenems, which are used to treat Extended-spectrum beta-lactamase (ESBL) producing bacteria. Here, we describe the development and evaluation of a DNA microarray for detection and genotyping of KPC genes (bla(KPC)) within 5 hours. To test the whole assay procedure (DNA extraction + DNA microarray assay) directly from clinical specimen, we compared two commercial DN...

  10. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA

    Barrett, Michael T; Scheffer, Alicia; Ben-Dor, Amir; Sampas, Nick; Lipson, Doron; Kincaid, Robert; Tsang, Peter; Curry, Bo; Baird, Kristin; Meltzer, Paul S.; Yakhini, Zohar; Bruhn, Laurakay; Laderman, Stephen

    2004-01-01

    Array-based comparative genomic hybridization (CGH) measures copy-number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders and for developing diagnostic and therapeutic targets. Arrays for CGH based on PCR products representing assemblies of BAC or cDNA clones typically require maintenance, propagation, replication, and verification of large clone sets. Furthermore, it is difficult to control the specificity of the hybridiz...

  11. An algorithm for automatic evaluation of the spot quality in two-color DNA microarray experiments

    Barillot Emmanuel

    2005-12-01

    Full Text Available Abstract Background Although DNA microarray technologies are very powerful for the simultaneous quantitative characterization of thousands of genes, the quality of the obtained experimental data is often far from ideal. The measured microarrays images represent a regular collection of spots, and the intensity of light at each spot is proportional to the DNA copy number or to the expression level of the gene whose DNA clone is spotted. Spot quality control is an essential part of microarray image analysis, which must be carried out at the level of individual spot identification. The problem is difficult to formalize due to the diversity of instrumental and biological factors that can influence the result. Results For each spot we estimate the ratio of measured fluorescence intensities revealing differential gene expression or change in DNA copy numbers between the test and control samples. We also define a set of quality characteristics and a model for combining these characteristics into an overall spot quality value. We have developed a training procedure to evaluate the contribution of each individual characteristic in the overall quality. This procedure uses information available from replicated spots, located in the same array or over a set of replicated arrays. It is assumed that unspoiled replicated spots must have very close ratios, whereas poor spots yield greater diversity in the obtained ratio estimates. Conclusion The developed procedure provides an automatic tool to quantify spot quality and to identify different types of spot deficiency occurring in DNA microarray technology. Quality values assigned to each spot can be used either to eliminate spots or to weight contribution of each ratio estimate in follow-up analysis procedures.

  12. Open-target sparse sensing of biological agents using DNA microarray

    Skaer Haley B

    2011-07-01

    Full Text Available Abstract Background Current biosensors are designed to target and react to specific nucleic acid sequences or structural epitopes. These 'target-specific' platforms require creation of new physical capture reagents when new organisms are targeted. An 'open-target' approach to DNA microarray biosensing is proposed and substantiated using laboratory generated data. The microarray consisted of 12,900 25 bp oligonucleotide capture probes derived from a statistical model trained on randomly selected genomic segments of pathogenic prokaryotic organisms. Open-target detection of organisms was accomplished using a reference library of hybridization patterns for three test organisms whose DNA sequences were not included in the design of the microarray probes. Results A multivariate mathematical model based on the partial least squares regression (PLSR was developed to detect the presence of three test organisms in mixed samples. When all 12,900 probes were used, the model correctly detected the signature of three test organisms in all mixed samples (mean(R2 = 0.76, CI = 0.95, with a 6% false positive rate. A sampling algorithm was then developed to sparsely sample the probe space for a minimal number of probes required to capture the hybridization imprints of the test organisms. The PLSR detection model was capable of correctly identifying the presence of the three test organisms in all mixed samples using only 47 probes (mean(R2 = 0.77, CI = 0.95 with nearly 100% specificity. Conclusions We conceived an 'open-target' approach to biosensing, and hypothesized that a relatively small, non-specifically designed, DNA microarray is capable of identifying the presence of multiple organisms in mixed samples. Coupled with a mathematical model applied to laboratory generated data, and sparse sampling of capture probes, the prototype microarray platform was able to capture the signature of each organism in all mixed samples with high sensitivity and specificity

  13. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

    Li, Yongjin

    2016-01-01

    The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use. PMID:26860568

  14. Optimization of cDNA microarrays procedures using criteria that do not rely on external standards

    Beisvag Vidar

    2007-10-01

    Full Text Available Abstract Background The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. Results We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. Conclusion The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly

  15. Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling

    Hansen, E.H.; Schembri, Mark; Klemm, Per; Schafer, T.; Molin, Søren; Gram, Lone

    2004-01-01

    A novel antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expr...... pinpoint one of the specific responses of E. coli-namely, the Cpx pathway, which is important for managing the stress response from the Curvularia haloperoxidase system.......A novel antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The...... was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to...

  16. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  17. DNA Microarray Analysis of Gene Expression in Antifungal Bacterium of Bacillus lenthmorbus WJ5

    This simultaneous expression levels of antifungal activity related was analyzed by DNA microarray. We constructured DNA chips contained 2,000 randomly digested genome spots of the antifungal bacterium of Bacillus lentimorbus WJ5 and compared it squantitative aspect with 7 antifungal activity deficient mutants induced by gamma radiation . From the analysis of microarray hybridization by the Gene Cluster, totally 408 genes were expressed and 20 genes among them were significantly suppressed in mutants. pbuX, ywbA, ptsG,yufO, and ftsY were simultaneously down-regulated in all muatants. It suggested that they were supposed to be related to the antifungal activity of B. lentimorbus WJ5

  18. Use of the cDNA microarray technology in the safety assessment of GM food plants

    Kok, E.J.; Kleter, G.A.; van Dijk

    2003-01-01

    This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates the potentials and applications of the techniques as a tool in safety assessment of genetically modified plants. The background for this evaluation of new techniques is that some concerns have been ra...

  19. Significance and statistical errors in the analysis of DNA microarray data

    Brody, James P.; Williams, Brian A.; Wold, Barbara J.; Quake, Stephen R

    2002-01-01

    DNA microarrays are important devices for high throughput measurements of gene expression, but no rational foundation has been established for understanding the sources of within-chip statistical error. We designed a specialized chip and protocol to investigate the distribution and magnitude of within-chip errors and discovered that, as expected from theoretical expectations, measurement errors follow a Lorentzian-like distribution, which explains the widely observed but unexplained ill-repro...

  20. Use of the cDNA microarray technology in thesafety assessment of GM food plants

    Pedersen, Jan W.; Knudsen, Ib; Eriksen, Folmer Damsted; Kärenlampi, S.; Mikalsen, A; Hammerling, U; Kok, E.; Bak, S.; Nielsen, Henrik Bjørn

    2003-01-01

    This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates the potentials and applications of the techniques as a tool in safety assessment of genetically modified plants. The background for this evaluation of new techniques is that some concerns have been ra...

  1. DNA Microarray Analysis of Chimpanzee Liver during Acute Resolving Hepatitis C Virus Infection

    Bigger, Catherine B.; Brasky, Kathleen M.; Lanford, Robert E.

    2001-01-01

    Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understoo...

  2. Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

    Regiane de Fátima Travensolo

    2009-06-01

    Full Text Available DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.

  3. Genome-wide screening for trait conferring genes using DNA microarrays

    Gill, R. T.; S. Wildt; Yang, Y. T.; Ziesman, S.; Stephanopoulos, G.

    2002-01-01

    We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with ...

  4. An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube® format

    Wiederanders B

    2006-06-01

    Full Text Available Abstract Background Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling, hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. Results In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube® format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR. Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA or In Vitro Transcription (IVT we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. Conclusion As the designed protocol for amplifying m

  5. Molecular characterization of Neisseria meningitidis isolates using a resequencing DNA microarray.

    Corless, Caroline E; Kaczmarski, Edward; Borrow, Ray; Guiver, Malcolm

    2008-05-01

    Neisseria meningitidis is a major cause of both meningitis and septicemia. Typically, isolates are characterized by using a combination of immunological phenotyping, using monoclonal and polyclonal antisera, and Sanger nucleotide sequencing of epitope-encoding variable regions, although these methods can be both time-consuming and limited by reagent availability. Herein, we describe and evaluate a novel microarray to define the porB and porA serotypes of N. meningitidis by the resequencing of variable regions in a single hybridization reaction. PCR products for each gene were amplified, pooled in equimolar concentrations, hybridized to the microarray, and analyzed using Affymetrix GeneChip DNA Analysis Software. Resequencing of the microarray data was then validated by comparison with sequencing data. Molecular profiles were generated for 50 isolates that were combinations of phenotypically typeable (ie, PorA and PorB) and non-typeable (PorB only) isolates. Microarray-generated profiles from isolates with a PorB phenotype were concordant with predicted profiles compared with a previously described typing scheme. In addition, 42% (8 of 19) of previously non-typeable samples were assigned a PorB type when tested using the microarray. The remaining isolates were novel types for which no typing antisera are currently available. The porA data were 97% concordant with Sanger nucleotide sequencing. These results suggest that that microarray resequencing may be a useful tool for the characterization of meningococci, particularly for those isolates that cannot be phenotyped, offering an alternative to conventional sequencing methods. PMID:18372424

  6. Design of a combinatorial dna microarray for protein-dnainteraction studies

    Mintseris, Julian; Eisen, Michael B.

    2006-07-07

    Background: Discovery of precise specificity oftranscription factors is an important step on the way to understandingthe complex mechanisms of gene regulation in eukaryotes. Recently,doublestranded protein-binding microarrays were developed as apotentially scalable approach to tackle transcription factor binding siteidentification. Results: Here we present an algorithmic approach toexperimental design of a microarray that allows for testing fullspecificity of a transcription factor binding to all possible DNA bindingsites of a given length, with optimally efficient use of the array. Thisdesign is universal, works for any factor that binds a sequence motif andis not species-specific. Furthermore, simulation results show that dataproduced with the designed arrays is easier to analyze and would resultin more precise identification of binding sites. Conclusion: In thisstudy, we present a design of a double stranded DNA microarray forprotein-DNA interaction studies and show that our algorithm allowsoptimally efficient use of the arrays for this purpose. We believe such adesign will prove useful for transcription factor binding siteidentification and other biological problems.

  7. Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray.

    Eom, Hyunsuk; Lee, Choul-Gyun; Jin, EonSeon

    2006-05-01

    The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3'-dihydroxy-beta, beta-carotene-4, 4'-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. PMID:16320067

  8. Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

    Kohama Chihiro

    2009-08-01

    Full Text Available Abstract Background Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs. NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. Results First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation

  9. Label-free DNA hybridization detection with molecular beacon immobilized in photopolymerized acrylamide gel microarray

    Wang, Hong; Li, Jiong; Liu, Quanjun; Liu, Heping; Lu, Zuhong; Zhu, Jijun

    2001-10-01

    Photopolymerized acrylamide gel microarray used to immobilize the molecular beacon for label free DNA hybridization detection is described in this paper. Polyacrylamide gel microarray was prepared by UV photopolymerization of 4% acrylamide in 40% glycerol, 0.002%methylene blue, 0.012% TEMED and 0.1M phosphate buffer (PHequals7) with a Relpel-silane pretreated quartz mask. This kind of three-dimensional gel microarray provides more than 100 times great immobilization capacity. The hybridization and other processes with it resemble a homogeneous liquid phase reaction rather than a heterogeneous liquid-solid interface reaction. The specially designed molecular beacons contain a 15 base loop sequence with 5 base pair stem, a 20 base thymine as spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the 20 base thymine is used to minimize destability caused by 5'-end immobilization. Confocal microscope was used to investigate the fluorescence intensity of gel immobilized molecular beacon probes. After hybridization we can easily distinguish complementary and noncomplementay targets with gel-immobilized molecular beacon probes. Image analysis showed that the fluorescence intensity ratio of complementary to noncomplementay probes is great than 5. The potential applications of gel-immobilized molecular beacon microarray are mutation detection, pathogenic detection, etc. in a parallel, cost saving and label-free detection way.

  10. Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

    Bordoni Roberta

    2007-11-01

    Full Text Available Abstract Background The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium. Results The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A; a growth slowdown until 52 h (Phase B; and another rapid growth phase from 56 h to 72 h (Phase C before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides that are clearly regulated in later phases. Conclusion The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional

  11. DNA-Microarrays zur Identifizierung von pathoadaptiven Mutationen und Antibiotikaresistenzen in extraintestinal pathogenen Escherichia coli (ExPEC)

    Barl, Timo

    2007-01-01

    Gegenstand dieser Arbeit war die Entwicklung von DNA-Microarrays zur Genotypisierung von E. coli für die klinische Diagnostik. Im ersten Teil der Dissertation wurde ein DNA-Microarray zur Detektion der häufigsten pathoadaptiven Mutationen in der Bindeuntereinheit von Typ 1-Fimbrien (FimH), die das Pathogenitätspotential von extraintestinal pathogenen Escherichia coli-Stämmen (ExPEC) erhöhen, entwickelt. Dieser Microarray wurde verwendet, um die fimH-Varianten von 131 E. coli-Isolaten zu genot...

  12. Analysis of Gene Expression Profile in Lung Adenosquamous Carcinoma Using cDNA Microarray

    YANG Fei; YANG Jiong; JIANG Man; YE Bo; ZHANG Yu-xia; CHEN Hong-lei; XIA Dong; LIU Ming-qiu

    2004-01-01

    Gene expression profile of the lung adenosquamous carcinoma was characterized by using cDNA microarray chip containing 4 096 human genes. Among target genes, 508 differentially expressed genes were identified in adenosquamous carcinoma of the lung, 232 genes were overexpressed and 276 genes were underexpressed. Among them, 92 genes are cell signals transduction genes, 34 genes are proto-oncogenes and tumor suppressor genes or cell cycle related genes or cell apoptosis related genes, 29 genes are cell skeleton genes, 28 genes are DNA synthesis, repair and recombination genes, 12 genes are DNA binding and transcription genes. These genes may be associated with the occurence and development of adenosquamous carinome of the lung.

  13. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  14. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Yu-Juan Xiang; Zhi-Gang Yu; Ming-Ming Guo; Qin-Ye Fu; Zhong-Bing Ma; De-Zong Gao; Qiang Zhang; Yu-Yang Li; Liang Li; Lu Liu; Chun-Miao Ye

    2015-01-01

    Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quanti-tatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  15. Use of the cDNA microarray technology in thesafety assessment of GM food plants

    Pedersen, Jan W.; Knudsen, Ib; Eriksen, Folmer Damsted;

    the potentials and applications of the techniques as a tool in safety assessment of genetically modified plants. The background for this evaluation of new techniques is that some concerns have been raised about the targeted approach used for evaluating the health risk of GM-plants. Today, an important......This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates...

  16. Significant overexpression of oligophrenin-1 in colorectal tumors detected by cDNA microarray analysis.

    Pinheiro, N A; Caballero, O L; Soares, F; Reis, L F; Simpson, A J

    2001-10-22

    The human oligophrenin-1 gene is ubiquitously expressed at low levels and expressed at high levels in the developing neuroepithelium of the neural tube. Mutations in this gene have been related to the X-linked mental retardation. Using cDNA microarrays, we found evidence that oligophrenin-1 is strongly up-regulated in colorectal tumors. Semiquantitative reverse transcriptase polymerase chain reaction confirmed this finding. Thus, a well-known nervous system-associated human gene transcript may also be an important colorectal tumor marker and potential therapeutic target. PMID:11595131

  17. Monitoring Gene Expression in Mixed Microbial Communities by Using DNA Microarrays

    Dennis, Philip; Edwards, Elizabeth A.; Liss, Steven N; Fulthorpe, Roberta

    2003-01-01

    A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed mic...

  18. Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

    Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

    2004-01-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., coul...

  19. Robust multi-scale clustering of large DNA microarray datasets with the consensus algorithm

    Grotkjær, Thomas; Winther, Ole; Regenberg, Birgitte; Nielsen, Jens; Hansen, Lars Kai

    2006-01-01

    Motivation: Hierarchical and relocation clustering (e.g. K-means and self-organizing maps) have been successful tools in the display and analysis of whole genome DNA microarray expression data. However, the results of hierarchical clustering are sensitive to outliers, and most relocation methods...... method is flexible and it is possible to find consensus clusters from different clustering algorithms. Thus, the algorithm can be used as a framework to test in a quantitative manner the homogeneity of different clustering algorithms. We compare the method with a number of state-of-the-art clustering...

  20. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten;

    2007-01-01

    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  1. Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays

    Gribble, Susan M; Ng, Bee Ling; Prigmore, Elena; Fitzgerald, Tomas; Carter, Nigel P

    2012-01-01

    Aarray painting is a technique that uses microarray technology to rapidly map chromosome translocation breakpoints. previous methods to map translocation breakpoints have used fluorescence in situ hybridization (FIsH) and have consequently been labor-intensive, time-consuming and restricted to the low breakpoint resolution imposed by the use of metaphase chromosomes. array painting combines the isolation of derivative chromosomes (chromosomes with translocations) and high-resolution microarray analysis to refine the genomic location of translocation breakpoints in a single experiment. In this protocol, we describe array painting by isolation of derivative chromosomes using a MoFlo flow sorter, amplification of these derivatives using whole-genome amplification and hybridization onto commercially available oligonucleotide microarrays. although the sorting of derivative chromosomes is a specialized procedure requiring sophisticated equipment, the amplification, labeling and hybridization of Dna is straightforward, robust and can be completed within 1 week. the protocol described produces good quality data; however, array painting is equally achievable using any combination of the available alternative methodologies for chromosome isolation, amplification and hybridization. PMID:19893508

  2. Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

    Pappas, Christopher T. [University of Wyoming, Laramie; Sram, Jakub [University of Wyoming, Laramie; Moskvin, Oleg V. [University of Wyoming, Laramie; Ivanov, Pavel S. [University of Wyoming, Laramie; Mackenzie, Christopher [University of Texas; Choudhary, Madhusudan [University of Texas; Land, Miriam L [ORNL; Larimer, Frank W [ORNL; Kaplan, Samuel [ORNL; Gomelsky, Mark [University of Wyoming, Laramie

    2004-07-01

    A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes-aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis-were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

  3. D-MaPs - DNA-microarray projects: web-based software for multi-platform microarray analysis

    Marcelo F. Carazzolle

    2009-01-01

    Full Text Available The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix. Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner. In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core.

  4. Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

    Hedegaard Jakob

    2009-07-01

    Full Text Available Abstract Background The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. Results Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. Conclusion It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experiment.

  5. Development of computational tools for the integrated analysis of DNA microarray data with applications in cancer research

    Valente, Eduardo

    2015-01-01

    The MAP-i Doctoral Program of the Universities of Minho, Aveiro and Porto DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests and predictive modeling, reducing the dat...

  6. Fabrication of polyurethane molecular stamps for the synthesis of DNA microarray

    Liu, Zhengchun; He, Quanguo; Xiao, Pengfeng; He, Nongyao; Lu, Zuhong; Bo, Liang

    2001-10-01

    Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.

  7. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray.

    González, Santiago F; Krug, Melissa J; Nielsen, Michael E; Santos, Ysabel; Call, Douglas R

    2004-04-01

    We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans. PMID:15070982

  8. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  9. DNA microarrays immobilized on unmodified plastics in a microfluidic biochip for rapid typing of Avian Influenza Virus

    Yi, Sun; Perch-Nielsen, Ivan R.; Dufva, Martin;

    2011-01-01

    directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on non-modified plastic surfaces. This one-step, cost-effective process provides very high immobilization and hybridization efficiencies and is applicable to many different types of polymers. Using this microarray fabrication technique, a...... portable cyclic olefin copolymer (COC) microarray device containing eight individually addressable microfluidic channels was developed for fast identification of Avian Influenza Virus (AIV) by DNA hybridization. This plastic biochip offers benefits of low fabrication cost and parallel processing of...

  10. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  11. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  12. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, 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; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  13. Normal uniform mixture differential gene expression detection for cDNA microarrays

    Raftery Adrian E

    2005-07-01

    Full Text Available Abstract Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002 1. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM, and Empirical Bayes for microarrays (EBarrays with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at http://www.bioconductor.org.

  14. Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

    Chen Wei; Fu Xiaobing; Sun Xiaoqing; Sun Tongzhu; Zhao Zhili; Yang Yinhui; Sheng Zhiyong

    2003-01-01

    Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.

  15. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Myers Connie

    2005-07-01

    Full Text Available Abstract Background Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Results Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN, was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect or intensity-dependent dye bias (referred later as intensity effect moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Conclusion Using LOOCV error of k-NNs as the

  16. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis.

    Sunwoo Chun

    Full Text Available A high phosphorus (HP diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus or a HP diet (containing 1.2% phosphorus. Gene Ontology analysis of differentially expressed genes (DEGs revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα, a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054 in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty

  17. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  18. An ensemble method for gene discovery based on DNA microarray data

    LI Xia; RAO Shaoqi; ZHANG Tianwen; GUO Zheng; ZHANG Qingpu; Kathy L. MOSER; Eric J. TOPOL

    2004-01-01

    The advent of DNA microarray technology has offered the promise of casting new insights onto deciphering secrets of life by monitoring activities of thousands of genes simultaneously.Current analyses of microarray data focus on precise classification of biological types,for example,tumor versus normal tissues.A further scientific challenging task is to extract disease-relevant genes from the bewildering amounts of raw data,which is one of the most critical themes in the post-genomic era,but it is generally ignored due to lack of an efficient approach.In this paper,we present a novel ensemble method for gene extraction that can be tailored to fulfill multiple biological tasks including(i)precise classification of biological types;(ii)disease gene mining; and(iii)target-driven gene networking.We also give a numerical application for(i)and(ii)using a public microarrary data set and set aside a separate paper to address(iii).

  19. Effects of aspirin on metastasis-associated gene expression detected by cDNA microarray

    Xue-qin GAO; Jin-xiang HAN; Hai-yan HUANG; Shi YAN; Chang-zheng SONG; Hai-nan HUANG

    2004-01-01

    AIM: To investigate the effect of aspirin on the metastasis-associated gene expression in 3AO ovarian cancer cells.METHODS: 3AO cells were treated with aspirin at the concentration of 1.2 mmol/L for 16 and 48 h, respectively.The total RNA was extracted with Trizol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction pathway molecules, adhesive molecules, growth factors and ESTs) fabricated in our lab. After normalization, the ratio of gene expression of aspirin treated to untreated 3AO cells being either 2 fold up higher or 0.5 fold down (lower) were defined as differential expression. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 4 genes were up-regulated and 14 genes were down-regulated in 3AO cells treated with aspirin for 16 h compared with untreated cells. While 24 genes were up-regulated and 10 genes were down-regulated in cells treated with aspirin for 48 h. Several up or down-regulated gene expression changes continued from 16 h to 48 h. CONCLUSION: Aspirin might exert its anti-metastasis effects on ovarian cancer by affecting metastasis-associated gene expression.

  20. Potential markers of tongue tumor progression selected by cDNA microarray.

    Carinci, F; Lo Muzio, L; Piattelli, A; Rubini, C; Chiesa, F; Ionna, F; Palmieri, A; Maiorano, E; Pastore, A; Laino, G; Dolci, M; Pezzetti, F

    2005-01-01

    Squamous cell carcinoma (SCC), the most frequent malignant tumor of the oral cavity, generally exhibits a poor prognosis and metastases are the main cause of death. This tumor often arises from pre-malignant lesions. To date, it is difficult to predict if and which pre-malignant lesions may progress into oral SCC using traditional methods. For these reasons, several studies are trying to identify markers useful in the progression of pre-malignant lesions and tumors. To define the genetic expression profile of tongue tumor progression we compared 9 dysplasias (DS), 8 tumors without metastasis (TWM), 11 metastasizing SCCs (MT) of the tongue, and a baseline of 11 normal tissues by using cDNA microarray containing 19.2 K clones. We initially applied hierarchical agglomerative clustering based on information from all 6026 clones. Results were obtained by performing a two steps analysis: a Significance Analysis of Microarray (SAM) and a Gene Ontology search. One hundred and five clones have statistically significant different expression levels (FDR ADAMTS2 and cathepsin O). Additionally, under-expressed genes encoded apoptosis-related proteins (PDCD4 and CASP4). In conclusion, we identified several genes differentially expressed in tumor progression which can potentially help in better classifying pre-malignant lesions and tongue SCCs. PMID:16164832

  1. Validation and characterization of DNA microarray gene expression data distribution and associated moments

    Chang Xiaoqing

    2010-11-01

    Full Text Available Abstract Background The data from DNA microarrays are increasingly being used in order to understand effects of different conditions, exposures or diseases on the modulation of the expression of various genes in a biological system. This knowledge is then further used in order to generate molecular mechanistic hypotheses for an organism when it is exposed to different conditions. Several different methods have been proposed to analyze these data under different distributional assumptions on gene expression. However, the empirical validation of these assumptions is lacking. Results Best fit hypotheses tests, moment-ratio diagrams and relationships between the different moments of the distribution of the gene expression was used to characterize the observed distributions. The data are obtained from the publicly available gene expression database, Gene Expression Omnibus (GEO to characterize the empirical distributions of gene expressions obtained under varying experimental situations each of which providing relatively large number of samples for hypothesis testing. All data were obtained from either of two microarray platforms - the commercial Affymetrix mouse 430.2 platform and a non-commercial Rosetta/Merck one. The data from each platform were preprocessed in the same manner. Conclusions The null hypotheses for goodness of fit for all considered univariate theoretical probability distributions (including the Normal distribution are rejected for more than 50% of probe sets on the Affymetrix microarray platform at a 95% confidence level, suggesting that under the tested conditions a priori assumption of any of these distributions across all probe sets is not valid. The pattern of null hypotheses rejection was different for the data from Rosetta/Merck platform with only around 20% of the probe sets failing the logistic distribution goodness-of-fit test. We find that there are statistically significant (at 95% confidence level based on the F

  2. A DNA microarray for the authentication of toxic traditional Chinese medicinal plants.

    Carles, Maria; Cheung, Matthew Kin; Moganti, Shanti; Dong, Tina T; Tsim, Karl W; Ip, Nancy Y; Sucher, Nikolaus J

    2005-06-01

    A silicon-based DNA microarray was designed and fabricated for the identification of toxic traditional Chinese medicinal plants. Species-specific oligonucleotide probes were derived from the 5S ribosomal RNA gene of Aconitum carmichaeli, A. kusnezoffi, Alocasia macrorrhiza, Croton tiglium, Datura inoxia, D. metel, D. tatula, Dysosma pleiantha, Dy. versipellis, Euphorbia kansui, Hyoscyamus niger, Pinellia cordata, P. pedatisecta, P. ternata, Rhododendron molle, Strychnos nux-vomica, Typhonium divaricatum and T. giganteum and the leucine transfer RNA gene of Aconitum pendulum and Stellera chamaejasme. The probes were immobilized via dithiol linkage on a silicon chip. Genomic target sequences were amplified and fluorescently labeled by asymmetric polymerase chain reaction. Multiple toxic plant species were identified by parallel genotyping. Chip-based authentication of medicinal plants may be useful as inexpensive and rapid tool for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals. PMID:15971136

  3. Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila

    Mariusz Żak

    2011-12-01

    Full Text Available Introduction:For the last five years, Legionella sp. infections and legionnaire’s disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations.Material/Methods:The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected.Results.We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain.Discussion:The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

  4. DNA microarray analysis of Staphylococcus aureus causing bloodstream infection: bacterial genes associated with mortality?

    Blomfeldt, A; Aamot, H V; Eskesen, A N; Monecke, S; White, R A; Leegaard, T M; Bjørnholt, J V

    2016-08-01

    Providing evidence for microbial genetic determinants' impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe-host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n = 305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n = 38) were characterised by DNA microarray analysis and spa typing. Fisher's exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing. PMID:27177754

  5. Dorsal horn-enriched genes identified by DNA microarray, in situ hybridization and immunohistochemistry

    Koblan Kenneth S

    2002-08-01

    Full Text Available Abstract Background Neurons in the dorsal spinal cord play important roles in nociception and pain. These neurons receive input from peripheral sensory neurons and then transmit the signals to the brain, as well as receive and integrate descending control signals from the brain. Many molecules important for pain transmission have been demonstrated to be localized to the dorsal horn of the spinal cord. Further understanding of the molecular interactions and signaling pathways in the dorsal horn neurons will require a better knowledge of the molecular neuroanatomy in the dorsal spinal cord. Results A large scale screening was conducted for genes with enriched expression in the dorsal spinal cord using DNA microarray and quantitative real-time PCR. In addition to genes known to be specifically expressed in the dorsal spinal cord, other neuropeptides, receptors, ion channels, and signaling molecules were also found enriched in the dorsal spinal cord. In situ hybridization and immunohistochemistry revealed the cellular expression of a subset of these genes. The regulation of a subset of the genes was also studied in the spinal nerve ligation (SNL neuropathic pain model. In general, we found that the genes that are enriched in the dorsal spinal cord were not among those found to be up-regulated in the spinal nerve ligation model of neuropathic pain. This study also provides a level of validation of the use of DNA microarrays in conjunction with our novel analysis algorithm (SAFER for the identification of differences in gene expression. Conclusion This study identified molecules that are enriched in the dorsal horn of the spinal cord and provided a molecular neuroanatomy in the spinal cord, which will aid in the understanding of the molecular mechanisms important in nociception and pain.

  6. DNA microarray unravels rapid changes in transcriptome of MK-801 treated rat brain

    Yuka; Kobayashi; Sofya; P; Kulikova; Junko; Shibato; Randeep; Rakwal; Hiroyuki; Satoh; Didier; Pinault; Yoshinori; Masuo

    2015-01-01

    AIM:To investigate the impact of MK-801 on gene expression patterns genome wide in rat brain regions. METHODS:Rats were treated with an intraperitoneal injection of MK-801 [0.08(low-dose) and 0.16(highdose) mg/kg] or NaC l(vehicle control). In a first series of experiment,the frontoparietal electrocorticogram was recorded 15 min before and 60 min after injection. In a second series of experiments,the whole brain of each animal was rapidly removed at 40 min post-injection,and different regions were separated:amygdala,cerebral cortex,hippocampus,hypothalamus,midbrain and ventral striatum on ice followed by DNA microarray(4 × 44 K whole rat genome chip) analysis.RESULTS:Spectral analysis revealed that a single systemic injection of MK-801 significantly and selectively augmented the power of baseline gamma frequency(30-80 Hz) oscillations in the frontoparietal electroencephalogram. DNA microarray analysis showed the largest number(up- and down- regulations) of gene expressions in the cerebral cortex(378),midbrain(376),hippocampus(375),ventral striatum(353),amygdala(301),and hypothalamus(201) under low-dose(0.08 mg/kg) of MK-801. Under high-dose(0.16 mg/kg),ventral striatum(811) showed the largest number of gene expression changes. Gene expression changes were functionally categorized to reveal expression of genes and function varies with each brain region.CONCLUSION:Acute MK-801 treatment increases synchrony of baseline gamma oscillations,and causes very early changes in gene expressions in six individual rat brain regions,a first report.

  7. Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset

    Reijmers, T.H.; Maliepaard, C.A.; Broeck, van den H.C.; Kessler, R.W.; Toonen, M.A.J.; Voet, van der H.

    2005-01-01

    Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A l

  8. Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system.

    Huguenin, Antoine; Moutte, Lauryane; Renois, Fanny; Leveque, Nicolas; Talmud, Deborah; Abely, Michel; Nguyen, Yohan; Carrat, Fabrice; Andreoletti, Laurent

    2012-06-01

    Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards. PMID:22499022

  9. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Wei Gong; Kun He; Mike Covington; S.R Dinesh-Kumar; Michael Snyder; Stacey L.Harmer; Yu-Xian Zhu; Xing Wang Deng

    2008-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to constructprotein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and proteinprotein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

  10. SiPM as miniaturised optical biosensor for DNA-microarray applications

    M.F. Santangelo

    2015-12-01

    Full Text Available A miniaturized optical biosensor for low-level fluorescence emitted by DNA strands labelled with CY5 is showed. Aim of this work is to demonstrate that a Si-based photodetector, having a low noise and a high sensitivity, can replace traditional detection systems in DNA-microarray applications. The photodetector used is a photomultiplier (SiPM, with 25 pixels. It exhibits a higher sensitivity than commercial optical readers and we experimentally found a detection limit for spotted dried samples of ∼1 nM. We measured the fluorescence signal in different operating conditions (angle of analysis, fluorophores concentrations, solution volumes and support. Once fixed the angle of analysis, for samples spotted on Al-TEOS slide dried, the system is proportional to the concentration of the analyte in the sample and is linear in the range 1 nM–1 μM. For solutions, the range of linearity ranges from 100 fM to 10 nM. The system potentialities and the device low costs suggest it as basic component for the design and fabrication of a cheap, easy and portable optical system.

  11. Gene-expression profiling of human mononuclear cells from welders using cDNA microarray.

    Rim, Kyung Taek; Park, Kun Koo; Kim, Yang Ho; Lee, Yong Hwan; Han, Jeong Hee; Chung, Yong Hyun; Yu, Il Je

    2007-08-01

    A toxicogenomic chip developed to detect welding-related diseases was tested and validated for field trials. To verify the suitability of the microarray, white blood cells (WBC) or whole blood was purified and characterized from 20 subjects in the control group (average work experience of 7 yr) and 20 welders in the welding-fume exposed group (welders with an average work experience of 23 yr). Two hundred and fifty-three rat genes homologous to human genes were obtained and spotted on the chip slide. Meanwhile, a human cDNA chip spotted with 8600 human genes was also used to detect any increased or decreased levels of gene expression among the welders. After comparing the levels of gene expression between the control and welder groups using the toxicogenomic chips, 103 genes were identified as likely to be specifically changed by welding-fume exposure. Eighteen of the 253 rat genes were specifically changed in the welders, while 103 genes from the human cDNA chip were specifically changed. The genes specifically expressed by the welders were associated with inflammatory responses, toxic chemical metabolism, stress proteins, transcription factors, and signal transduction. In contrast, there was no significant change in the genes related to short-term welding-fume exposure, such as tumor necrosis factor (TNF)-alpha and interleukin. In conclusion, if further validation studies are conducted, the present toxicogenomic gene chips could be used for the effective monitoring of welding-fume-exposure-related diseases among welders. PMID:17654244

  12. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  13. The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

    Pesta David

    2003-06-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

  14. Rapid quantification and taxonomic classification of environmentalDNA from both prokaryotic and eukaryotic origins using a microarray

    DeSantis, Todd Z.; Stone, Carol E.; Murray, Sonya R.; Moberg,Jordan P.; Andersen, Gary L.

    2005-02-22

    A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations (r=0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales, whereas a vigorous disruption produced peak intensities for Vibrionales,Clostridiales, and Bacillales.

  15. Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

    Gravelat Fabrice

    2010-09-01

    Full Text Available Abstract Background Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes. Results First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality can be used to study a complex environment efficiently. Conclusions We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments

  16. 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

    Carr Steven M; Flynn Sarah MC

    2007-01-01

    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 se...

  17. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  18. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10−16 mol L−1. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10−16 mol L−1. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of c

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

    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.

  20. Monitoring the Expression Profiles of Cereal Crops Seedlings by Using Rice cDNA Microarray

    SUN Liang-xian; DONG Hai-tao; LI De-bao

    2004-01-01

    Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3 311 unique rice transcripts (including 1 639 endosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of 1-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [α-33p] labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cereal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6 %) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings while considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profiles, 84 genes were identified constantly expressed in all the four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.

  1. Monitoring the Expression Profiles of Cereal Crops Seedlings by Using Rice cDNA Microarray

    SUNLiang-xian; DONGHai-tao; LIDe-bao

    2004-01-01

    Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3311 unique rice transcripts (including 1 639 cndosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of l-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [P] labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cereal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6%) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings whilc considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profdes, 84 genes were identified constantly expressed in all the four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.

  2. DNA microarray analysis of Salmonella serotype Typhimurium strains causing different symptoms of disease

    Helms Morten

    2010-03-01

    Full Text Available Abstract Background Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest. Results Strains were selected from patients with mild infections (n = 9 and patients with severe infections (n = 9 and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3 were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection. Conclusions We investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

  3. Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray1

    PING-PING ZHOU; JIAN-ZHONG ZHANG; YUAN-HAI YOU; YONG-NING WU

    2008-01-01

    Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified(GM)soybean.Seventeen capture probes(PCR products)and 17 pairs of corresponding primers were designed according to the genetic characteristies of Rroundup Ready soybean(GTS40-3-2),maize (Mort810,Nk603,GA21),canola(T45,MS1/RF1),and rice(SCK)in many identified GM crops.All of the probes were categorized and identified as species-specific probes.One negative probe and one positive control probe were uscd to assess the efficiency of all reactions,and therefore eliminate any false positive and negative results.After multiplex PCR reaction,amplicons were adulterated with Cy5-dUTP and hvbridized with DNA microarray.The array was then scanned to display the specific hybridization signals of target genes.The assay was applied to the analysis of sample of ccrtified transgenic soybean (Roundup Ready GTS40-3-2)and canola(MS1/RF1). Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability.Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events,indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

  4. Establishment of cDNA Microarray Analysis at the Genomic Medicine Research Core Laboratory (GMRCL) of Chang Gung Memorial Hospital .

    Tzu-Hao Wang; Yun-Shien Lee; En-Shih Chen; Wei-Hsiang Kong; Lung-Kun Chen; Ding-Wei Hsueh; Min-Li Wei; Hsing-Shih Wang; Ying-Shiung Lee

    2004-01-01

    Background: Advances in molecular and computational biology have led to the developmentof powerful, high-throughput methods for analysis of differential geneexpression, which are opening up new opportunities in genomic medicine.DNA microarray technology has been enthusiastically integrated into basicbiomedical research and will eventually become a molecular monitoring toolfor various clinical courses.Methods: As a core research facility of Chang Gung University (CGU) and ChangGung Memorial Ho...

  5. FSL-based Hardware Implementation for Parallel Computation of cDNA Microarray Image Segmentation

    Bogdan Bot; Simina Emerich; Sorin Martoiu; Bogdan Belean

    2015-01-01

    The present paper proposes a FPGA based hardware implementations for microarray image processing algorithms in order eliminate the shortcomings of the existing software platforms: user intervention, increased computation time and cost. The proposed image processing algorithms exclude user intervention from processing. An application-specific architecture is designed aiming microarray image processing algorithms parallelization in order to speed up computation. Hardware architectures for logar...

  6. Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells

    Ahmed Sharlin

    2004-07-01

    Full Text Available Abstract Background cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. Results We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM. Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values Conclusions We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (

  7. Microarrays, Integrated Analytical Systems

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  8. Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

    Boermans Herman J

    2006-11-01

    Full Text Available Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR. Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

  9. Rapid virological diagnosis of central nervous system infections by use of a multiplex reverse transcription-PCR DNA microarray.

    Leveque, Nicolas; Van Haecke, Adrien; Renois, Fanny; Boutolleau, David; Talmud, Deborah; Andreoletti, Laurent

    2011-11-01

    Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS. PMID:21918017

  10. Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses.

    Amundson, S A; Bittner, M; Chen, Y; Trent, J; Meltzer, P; Fornace, A J

    1999-06-17

    The fate of cells exposed to ionizing radiation (IR) may depend greatly on changes in gene expression, so that an improved view of gene induction profiles is important for understanding mechanisms of checkpoint control, repair and cell death following such exposures. We have used a quantitative fluorescent cDNA microarray hybridization approach to identify genes regulated in response to 7-irradiation in the p53 wild-type ML-1 human myeloid cell line. Hybridization of the array to fluorescently-labeled RNA from treated and untreated cells was followed by computer analysis to derive relative changes in expression levels of the genes present in the array, which agreed well with actual quantitative changes in expression. Forty-eight sequences, 30 not previously identified as IR-responsive, were significantly regulated by IR. Induction by IR and other stresses of a subset of these genes, including the previously characterized CIP1/ WAF1, MDM2 and BAX genes, as well as nine genes not previously reported to be IR-responsive, was examined in a panel of 12 human cell lines. Responses varied widely in cell lines with different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses. Two of the newly identified IR-responsive genes, FRA-1 and ATF3, showed a p53-associated component to their IR-induction, and this was confirmed both in isogenic human cell lines and in mouse thymus. The majority of the IR-responsive genes, however, showed no indication of p53-dependent regulation, representing a potentially important class of stress-responsive genes in leukemic cells. PMID:10380890

  11. DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness

    Feldman Mark

    2008-12-01

    Full Text Available Abstract Background A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms. Results Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated. As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR. Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments. Conclusion These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.

  12. DNA microarray analysis of fluconazole resistance in a laboratory Candida albicans strain

    2008-01-01

    Several mechanisms are responsible for the acquired fluconazole (FLC) resistance in Candida albicans. In this study, we developed a FLC-resistant C. albicans strain through serial cultures of a FLC-susceptible C. albicans strain with inhibitory concentrations of FLC. Complimen-tary DNA microarray analysis and real-time reverse tran-scription-polymerase chain reaction were used to investi-gate gene expression changes during the acquisition of azole resistance in the susceptible parental strain and the resis-tant daughter strain. The differentially expressed genes rep-resented functions as diverse as transporters (e.g. CDRI, PDR17), ergosterol biosynthesis (e.g. ERG2, ERG9), sterol metabolism (e.g. ARE2, IPF6464), energy metabolism (e.g. ADH3, AOX2) and transcription factors (e.g. FCR1, ECM22). Functional analysis revealed that energy-depen-dent efflux activity of membrane transporters increased and that ergosterol content decreased with the accumulation of sterol intermediates in the resistant strain as compared with the susceptible strain. We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hy-peractivity of Tac1 could be the reason for overexpression of CDR1, CDR2, and PDR17 in the resistant strain.Furthermore, a single amino acid difference (DI9E) in ERG3 that led to the inactivation of Erg3 could account for both sterol precursor accumulation and the changes in the ex-pression of ergosterol biosynthesis genes in this resistant strain. These findings expand the understanding of poten-tial novel molecular targets of FLC resistance in clinical C.albicans isolates.

  13. Genome-wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases

    Harris, R Alan; Nagy-Szakal, Dorottya; Pedersen, Natalia; Opekun, Antone; Bronsky, Jiri; Munkholm, Pia; Jespersgaard, Cathrine; Andersen, PaalSkytt; Melegh, Bela; Ferry, George; Jess, Tine; Kellermayer, Richard

    2012-01-01

    Crohn's disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation of ...... cytosines within CpG dinucleotides) is an epigenetic modification, which can respond to environmental influences. We investigated whether DNA methylation might be connected with IBD in peripheral blood leukocyte (PBL) DNA by utilizing genome-wide microarrays.......Crohn's disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation of...

  14. Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays

    Hedegaard, Jakob; Skovgaard, Kerstin; Mortensen, Shila;

    2007-01-01

    inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver. Conclusion: The obtained results are......-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and...... tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products. Results: A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus...

  15. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    Hai-Tao Wang; Jian-Ping Kong; Fang Ding; Xiu-Qin Wang; Ming-Rong Wang; Lian-Xin Liu; Min Wu; Zhi-Hua Liu

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.METHODS: The authors first constructed pcDNA3.1/mychis expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes,classification was performed according to their function and cellular component.RESULTS: Human EMP-1 gene can be stably expressed in ECg706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved incell signaling, cell communication and adhesion regulators.

  16. Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies.

    Soanes, Kelly H; Achenbach, John C; Burton, Ian W; Hui, Joseph P M; Penny, Susanne L; Karakach, Tobias K

    2011-11-01

    One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other

  17. Hypoxia-induced regulation of MAPK phosphatase-1 as identified by subtractive suppression hybridization and cDNA microarray analysis.

    Seta, K A; Kim, R; Kim, H W; Millhorn, D E; Beitner-Johnson, D

    2001-11-30

    Subtractive suppression hybridization was used to generate a cDNA library enriched in cDNA sequences corresponding to mRNA species that are specifically up-regulated by hypoxia (6 h, 1% O(2)) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPK phosphatase-1 (MKP-1) was highly represented in this library. Clones were arrayed on glass slides to create a hypoxia-specific cDNA microarray chip. Microarray, northern blot, and western blot analyses confirmed that MKP-1 mRNA and protein levels were up-regulated by hypoxia by approximately 8-fold. The magnitude of the effect of hypoxia on MKP-1 was approximately equal to that induced by KCl depolarization and much larger than the effects of either epidermal growth factor or nerve growth factor on MKP-1 mRNA levels. In contrast to the calcium-dependent induction of MKP-1 by KCl depolarization, the effect of hypoxia on MKP-1 persisted under calcium-free conditions. Cobalt and deferoxamine also increased MKP-1 mRNA levels, suggesting that hypoxia-inducible factor proteins may play a role in the regulation of MKP-1 by hypoxia. Pretreatment of cells with SB203580, which inhibits p38 kinase activity, significantly reduced the hypoxia-induced increase in MKP-1 RNA levels. Thus, hypoxia robustly increases MKP-1 levels, at least in part through a p38 kinase-mediated mechanism. PMID:11577072

  18. CRISPR adaptation biases explain preference for acquisition of foreign DNA

    Levy, Asaf; Goren, Moran G.; Yosef, Ido; Auster, Oren; Manor, Miriam; Amitai, Gil; Edgar, Rotem; Qimron, Udi; Sorek, Rotem

    2015-01-01

    In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are...

  19. The Gene Expression Profile of D-galactose Induced Aging Model Rat Using cDNA Microarray

    Li Min(李珉); Wang Gang; Zhang Wei; Wang Miqu; Zhang Yizheng

    2004-01-01

    In order to study the molecular mechanism of D-galactose induced aging model, cDNA microarray is used to analyze gene expression profiles of both normal and D-galactose induced aging model rats. D-galactose induced aging model rats are injected with D-galactose, while normal rats are injected with physiological saline as control. After 7 weeks, the two groups of rats are killed simultaneously. Their livers are harvested for genome-wide expression analysis. D-galactose treated rats showed changes in gene expression associated with increase or decrease in xenobiotic metabolism, protein metabolism and energy metabolism.

  20. Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

    ZENG Yue-bin; QIAN Yuan-shu; MA Lian; GU Hong-ni

    2007-01-01

    Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1),genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump

  1. Identification of hypoxia-responsive genes in a dopaminergic cell line by subtractive cDNA libraries and microarray analysis.

    Beitner-Johnson, D; Seta, K; Yuan, Y; Kim, H -W.; Rust, R T.; Conrad, P W.; Kobayashi, S; Millhorn, D E.

    2001-07-01

    Transplantation of dopamine-secreting cells harvested from fetal mesencephalon directly into the striatum has had limited success as a therapy for Parkinson's disease. A major problem is that the majority of the cells die during the first 3 weeks following transplantation. Hypoxia in the tissue surrounding the graft is a potential cause of the cell death. We have used subtractive cDNA libraries and microarray analysis to identify the gene expression profile that regulates tolerance to hypoxia. An improved understanding of the molecular basis of hypoxia-tolerance may allow investigators to engineer cells that can survive in the hypoxic environment of the brain parenchyma following transplantation. PMID:11331199

  2. A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis

    Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

    2016-01-01

    To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer. PMID:26966393

  3. Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

    Utsumi, Yoshinori; Tanaka, Maho; Kurotani, Atsushi; Yoshida, Takuhiro; Mochida, Keiichi; Matsui, Akihiro; Ishitani, Manabu; Sraphet, Supajit; Whankaew, Sukhuman; Asvarak, Thipa; Narangajavana, Jarunya; Triwitayakorn, Kanokporn; Sakurai, Tetsuya; Seki, Motoaki

    2016-07-01

    Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants. PMID:27138000

  4. Supervised Learning in Adaptive DNA Strand Displacement Networks.

    Lakin, Matthew R; Stefanovic, Darko

    2016-08-19

    The development of engineered biochemical circuits that exhibit adaptive behavior is a key goal of synthetic biology and molecular computing. Such circuits could be used for long-term monitoring and control of biochemical systems, for instance, to prevent disease or to enable the development of artificial life. In this article, we present a framework for developing adaptive molecular circuits using buffered DNA strand displacement networks, which extend existing DNA strand displacement circuit architectures to enable straightforward storage and modification of behavioral parameters. As a proof of concept, we use this framework to design and simulate a DNA circuit for supervised learning of a class of linear functions by stochastic gradient descent. This work highlights the potential of buffered DNA strand displacement as a powerful circuit architecture for implementing adaptive molecular systems. PMID:27111037

  5. Selective Discrimination of Listeria monocytogenes Epidemic Strains by a Mixed-Genome DNA Microarray Compared to Discrimination by Pulsed-Field Gel Electrophoresis, Ribotyping, and Multilocus Sequence Typing

    Borucki, Monica K.; Kim, So Hyun; Call, Douglas R.; Smole, Sandra C.; Pagotto, Franco

    2004-01-01

    Listeria monocytogenes can cause serious illness in humans, and subsequent epidemiological investigation requires molecular characterization to allow the identification of specific isolates. L. monocytogenes is usually characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping. DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE and ribotyping, microarrays can be used to identify specific g...

  6. Resolution of large and small differences in gene expression using models for the Bayesian analysis of gene expression levels and spotted DNA microarrays

    Townsend Jeffrey P

    2004-01-01

    Abstract Background The detection of small yet statistically significant differences in gene expression in spotted DNA microarray studies is an ongoing challenge. Meeting this challenge requires careful examination of the performance of a range of statistical models, as well as empirical examination of the effect of replication on the power to resolve these differences. Results New models are derived and software is developed for the analysis of microarray ratio data. These models incorporate...

  7. Functionalization of Poly- (methyl methacrylate) (PMMA) as a substrate for DNA microarrays

    Fixe, A.F.; Dufva, Hans Martin; Telleman, Pieter; Christensen, Claus Bo Vöge

    2004-01-01

    differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray(TM) slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm(2), respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable...

  8. Cell cycle control after DNA damage: arrest, recovery and adaptation

    DNA damage triggers surveillance mechanisms, the DNA checkpoints, that control the genome integrity. The DNA checkpoints induce several responses, either cellular or transcriptional, that favor DNA repair. In particular, activation of the DNA checkpoints inhibits cell cycle progression in all phases, depending on the stage when lesions occur. These arrests are generally transient and cells ultimately reenter the cell division cycle whether lesions have been repaired (this process is termed 'recovery') or have proved un-repairable (this option is called 'adaptation'). The mechanisms controlling cell cycle arrests, recovery and adaptation are largely conserved among eukaryotes, and much information is now available for the yeast Saccharomyces cerevisiae, that is used as a model organism in these studies. (author)

  9. Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

    Moran, Sebastián; Vizoso, Miguel; Martinez-Cardús, Anna; Gomez, Antonio; Matías-Guiu, Xavier; Chiavenna, Sebastián M; Fernandez, Andrés G; Esteller, Manel

    2014-01-01

    A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethyl...

  10. Genome-wide estimation of transcript concentrations from spotted cDNA microarray data

    Frigessi, Arnoldo; van de Wiel, Mark A; Holden, Marit; Svendsrud, Debbie H; Glad, Ingrid K; Lyng, Heidi

    2005-01-01

    A method providing absolute transcript concentrations from spotted microarray intensity data is presented. Number of transcripts per µg total RNA, mRNA or per cell, are obtained for each gene, enabling comparisons of transcript levels within and between tissues. The method is based on Bayesian statistical modelling incorporating available information about the experiment from target preparation to image analysis, leading to realistically large confidence intervals for estimated concentrations...

  11. Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins

    Schoenberg Fejzo, Marlena; Slamon, Dennis J.

    2001-01-01

    Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified thi...

  12. Using DNA Microarrays To Identify Library-Independent Markers for Bacterial Source Tracking

    Soule, Marilyn; Kuhn, Edward; Loge, Frank; Gay, John; Call, Douglas R.

    2006-01-01

    Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterf...

  13. Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays

    Bendixen Christian

    2007-04-01

    Full Text Available Abstract Background The bacterium Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after A. pleuropneumoniae infection is still not clarified. The objective of this study was hence to characterise the transcriptional response, measured by using cDNA microarrays, in pigs 24 hours after experimental inoculation with A. pleuropneumoniae. Methods Microarray analyses were conducted to reveal genes being differentially expressed in inflamed versus non-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products. Results A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver. Conclusion The obtained results are largely in accordance with previous studies of the mammalian immune response. Furthermore, a number of differentially expressed genes have not previously been associated

  14. Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.

    Geue, Lutz; Stieber, Bettina; Monecke, Stefan; Engelmann, Ines; Gunzer, Florian; Slickers, Peter; Braun, Sascha D; Ehricht, Ralf

    2014-08-01

    In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise. PMID:24899022

  15. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    Feng Yan; Xiao-Min Wang

    2007-01-01

    AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension.METHODS: The Biostar-H140s chip containing 14112 spots of cDNAs were used to investigate the difference of the expression. The total RNA extracted from macrophages isolated from both normal spleen and portal hypertensive spleen was reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5) labeled dCTP to prepare the hybridization probes.After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. That was repeated three times,and only the genes which had differential expression in all three chips were considered to be associated with hypersplenism in portal hypertension.RESULTS: Eight hundred and ninety-six, 1330 and 898 genes were identified to be differentially expressed in three chips, respectively. One hundred and twenty-one genes (0.86%) were identified to be differentially expressed in all three chips, including 21 up-regulated genes and 73 down-regulated genes. The differentially expressed genes were related to ionic channel and transport protein, cyclin, cytoskeleton, cell receptor, cell signal conduct, metabolism, immune, and so on. These genes might be related to the hypersplenism in portal hypertension.CONCLUSION: The investigations based on cDNA microarray can screen differentially expressed genes of macrophages between normal spleen and portal hypertensive spleen, thus may provide a new idea in studying the pathogenesis of hypersplenism in portal hypertension.

  16. Resolving individuals contributing trace amounts of DNA to highly complex mixtures using high-density SNP genotyping microarrays.

    Nils Homer

    2008-08-01

    Full Text Available We use high-density single nucleotide polymorphism (SNP genotyping microarrays to demonstrate the ability to accurately and robustly determine whether individuals are in a complex genomic DNA mixture. We first develop a theoretical framework for detecting an individual's presence within a mixture, then show, through simulations, the limits associated with our method, and finally demonstrate experimentally the identification of the presence of genomic DNA of specific individuals within a series of highly complex genomic mixtures, including mixtures where an individual contributes less than 0.1% of the total genomic DNA. These findings shift the perceived utility of SNPs for identifying individual trace contributors within a forensics mixture, and suggest future research efforts into assessing the viability of previously sub-optimal DNA sources due to sample contamination. These findings also suggest that composite statistics across cohorts, such as allele frequency or genotype counts, do not mask identity within genome-wide association studies. The implications of these findings are discussed.

  17. Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252Cf neutron and X-rays

    Yang Zhen-Zhou

    2010-02-01

    Full Text Available Abstract Background The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to 252Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes. Methods HeLa cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR. Results The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to 252Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated. Conclusions Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these "radioresistance" genes will lead to new therapeutic targets for cervical cancer.

  18. Incorporating feature ranking and evolutionary methods for the classification of high-dimensional DNA microarray gene expression data

    Mani Abedini

    2013-05-01

    Full Text Available AbstractBackgroundDNA microarray gene expression classification poses a challenging task to the machine learning domain. Typically, the dimensionality of gene expression data sets could go from several thousands to over 10,000 genes. A potential solution to this issue is using feature selection to reduce the dimensionality.AimThe aim of this paper is to investigate how we can use feature quality information to improve the precision of microarray gene expression classification tasks. Method We propose two evolutionary machine learning models based on the eXtended Classifier System (XCS and a typical feature selection methodology. The first one, which we call FS-XCS, uses feature selection for feature reduction purposes. The second model is GRD-XCS, which uses feature ranking to bias the rule discovery process of XCS.ResultsThe results indicate that the use of feature selection / ranking methods is essential for tackling high-dimensional classification tasks, such as microarray gene expression classification. However, the results also suggest that using feature ranking to bias the rule discovery process performs significantly better than using the feature reduction method. In other words, using feature quality information to develop a smarter learning procedure is more efficient than reducing the feature set. ConclusionOur findings have shown that extracting feature quality information can assist the learning process and improve classification accuracy. On the other hand, relying exclusively on the feature quality information might potentially decrease the classification performance (e.g., using feature reduction. Therefore, we recommend a hybrid approach that uses feature quality information to direct the learning process by highlighting the more informative features, but at the same time not restricting the learning process to explore other features.

  19. Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

    Done Susan J

    2011-06-01

    Full Text Available Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status. Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated

  20. Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

    The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. We

  1. Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk.

    Mogi, Takeyuki; Hatakeyama, Keiichi; Taguchi, Tomoyuki; Wake, Hitoshi; Tanaami, Takeo; Hosokawa, Masahito; Tanaka, Tsuyoshi; Matsunaga, Tadashi

    2011-01-15

    This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader™) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader™ consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader™ with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader™ for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis. PMID:20951567

  2. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse.

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2016-03-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood-brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

  3. Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

    Liebe, Sebastian; Christ, Daniela S; Ehricht, Ralf; Varrelmann, Mark

    2016-01-01

    Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present. PMID:26524545

  4. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

    Jang Soo Yook

    2016-03-01

    Full Text Available Naturally occurring astaxantin (ASX is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses using DNA microarray (Agilent 4 × 44 K whole mouse genome chip analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197 as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus.

  5. Feasibility study on blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies. Final report

    The final report on the feasibility of blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies covers the following topics: blood samples; methodologies: 2D gel electrophoresis; protein identification using MALDI-MS; accomplishment and evaluation of the proteomics and cDNA microarray analysis.

  6. cDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

    Purpose: To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. Methods and Materials: Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Results: We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor κB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. Conclusions: Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another 'key limitation' is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.

  7. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Nicolas M Bertagnolli

    Full Text Available To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  8. cDNA microarray analysis of rat alveolar epithelial cells following exposure to organic extract of diesel exhaust particles

    Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the mechanisms underlying the effects of air pollutants including DEP on lung diseases. Using a cDNA microarray, we examined changes in gene expression in SV40T2 cells, a rat alveolar type II epithelial cell line, following exposure to an organic extract of DEP. We identified candidate sensitive genes that were up- or down-regulated in response to DEP. The cDNA microarray analysis revealed that a 6-h exposure to the DEP extract (30 μg/ml) increased (>2-fold) the expression of 51 genes associated with drug metabolism, antioxidation, cell cycle/proliferation/apoptosis, coagulation/fibrinolysis, and expressed sequence tags (ESTs), and decreased (<0.5-fold) that of 20 genes. In the present study, heme oxygenase (HO)-1, an antioxidative enzyme, showed the maximum increase in gene expression; and type II transglutaminase (TGM-2), a regulator of coagulation, showed the most prominent decrease among the genes. We confirmed the change in the HO-1 protein level by Western blot analysis and that in the enzyme activity of TGM-2. The organic extract of DEP increased the expression of HO-1 protein and decreased the enzyme activity of TGM-2. Furthermore, these effects of DEP on either HO-1 or TGM-2 were reduced by N-acetyl-L-cysteine (NAC), thus suggesting that oxidative stress caused by this organic fraction of DEP may have induced these cellular responses. Therefore, an increase in HO-1 and a decrease in TGM-2 might be good markers of the biological response to organic compounds of airborne particulate substances

  9. Enhancing the Sensitivity of DNA Microarray Using Dye-Doped Silica Nanoparticles: Detection of Human Papilloma Virus

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Canton, G.; Cretaio, E.

    2010-10-01

    DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is based on the specific hybridization between probe sequences deposited in array and a target ss-DNA amplified by PCR and functionalized by a fluorescent dye. Organic labels have well known disadvantages like photobleaching and low signal intensities, which put a limitation to the lower amount of DNA material that can be detected. Therefore for trace analysis the development of more efficient biomarkers is required. With this aim we present in this paper the synthesis and application of alternative hybrid nanosystems obtained by incorporating standard fluorescent molecules into monodisperse silica nanoparticles. Efficient application to the detection of Human Papilloma Virus is demonstrated. This virus is associated to the formation of cervical cancer, a leading cause of death by cancer for women worldwide. It is shown that the use of the novel biomarkers increases the optical signal of about one order of magnitude with respect to the free dyes or quantum dots in conventional instruments. This is due to the high number of molecules that can be accommodated into each nanoparticle, to the reduced photobleaching and to the improved environmental protection of the dyes when encapsulated in the silica matrix. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalizability and bio-compatibility make them very promising for present and future bio-labeling and bio-imaging applications.

  10. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    McNicholas, Sinead

    2012-02-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

  11. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    McNicholas, Sinead

    2011-12-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

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

    Skov, Vibe

    2007-01-01

    Polycystic ovary syndrome (PCOS) affects 5-10 % of women during their reproductive age and the incidence of the disease is increasing worldwide. More than 50 % of women with PCOS are insulin resistant leading to an increased risk of type 2 diabetes (T2D) and cardiovascular diseases. However......, the molecular mechanisms of insulin resistance in skeletal muscle of women with PCOS are largely unknown. The aims of the Ph.D. thesis were: to identify biological pathways of importance for insulin resistance in skeletal muscle in a group of insulin resistant obese PCOS patients using global pathway analysis...... (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...

  13. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA-nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive

  14. A Condition-Enumeration Tree method for mining biclusters from DNA microarray data sets.

    Chen, Jiun-Rung; Chang, Ye-In

    2009-07-01

    Biclustering, which performs simultaneous clustering of rows (e.g., genes) and columns (e.g., conditions), has proved of great value for finding interesting patterns from microarray data. To find biclusters, a model called pCluster was proposed. A pCluster consists of a set of genes and a set of conditions, where the expression levels of these genes have a similar variation under these conditions. Based on this model, most of the previous methods need to compute MDSs (maximum dimension sets) for every two genes in the microarray data. Since the number of genes is far larger than the number of conditions, this step is inefficient. Another method called MicroCluster was proposed. This method does not compute MDSs for every two genes, and transforms the problem into a graph problem. However, it needs to solve the Maximal Clique problem, which is NP-Complete. To avoid the above disadvantages, in this paper, we propose a new method, CE-Tree (Condition-Enumeration Tree), for finding pClusters. Instead of generating MDSs for every two genes, we generate only MDSs for every two conditions. Then, based only on these MDSs, we expand the CE-Tree in a special local breadth-first within global depth-first manner to efficiently find all pClusters. We also utilize the idea of the traditional hash join approach to efficiently support the CE-Tree. From the simulation results, we show that the CE-Tree method could find pClusters more efficiently than those previous methods. PMID:19393714

  15. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

    Weingeist, David M.; Ge, Jing; Wood, David K.; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A.; Yaffe, Michael B.; Floyd, Scott; Engelward, Bevin P.

    2013-01-01

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluatio...

  16. Monitoring expression profiles of rice (Oryza sativa L.) genes under abiotic stresses using cDNA Microarray Analysis (abstract)

    Transcript regulation in response to cold, drought, high salinity and ABA application was investigated in rice (Oryza sativa L., Nipponbare) with microarray analysis including approx. 1700 independent DNA elements derived from three cDNA libraries constructed from 15-day old rice seedlings stressed with drought, cold and high salinity. A total of 141 non-redundant genes were identified, whose expression ratios were more than three-fold compared with the control genes for at least one of stress treatments in microarray analysis. However, after RNA gel blot analysis, a total of 73 genes were identified, among them the transcripts of 36, 62, 57 and 43 genes were found increased after cold, drought, high salinity and ABA application, respectively. Sixteen of these identified genes have been reported previously to be stress inducible in rice, while 57 of which are novel that have not been reported earlier as stress responsive in rice. We observed a strong association in the expression patterns of stress responsive genes and found 15 stress inducible genes that responded to all four treatments. Based on Venn diagram analysis, 56 genes were induced by both drought and high salinity, whereas 22 genes were upregulated by both cold and high salinity stress. Similarly 43 genes were induced by both drought stress and ABA application, while only 17 genes were identified as cold and ABA inducible genes. These results indicated the existence of greater cross talk between drought, ABA and high salinity stress signaling processes than those between cold and ABA, and cold and high salinity stress signaling pathways. The cold, drought, high salinity and ABA inducible genes were classified into four gene groups from their expression profiles. Analysis of data enabled us to identify a number of promoters and possible cis-acting DNA elements of several genes induced by a variety of abiotic stresses by combining expression data with genomic sequence data of rice. Comparative analysis of

  17. Gene expression profiling in Barrett's esophagus and cardia intestinal metaplasia:A comparative analysis using cDNA microarray

    Ying Chang; Jun Gong; Bin Liu; Jun Zhang; Fei Dai

    2004-01-01

    AIM: To study the difference of gene expression profile changes in Barrett's esophagus (BE) and cardia intestinal metaplasia (CIM) and to screen the novel genes in the early stage by cDNA microarray.METHODS: cDNA retrotranscribed from an equal amount of mRNA from BE and CIM epithelial tissues was labeled with Cy3 and Cy5 fluorescence as probes. The mixed probe was hybridized with three pieces of BiostarH-40 s double dot human whole gene chip. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 3.0 software.RESULTS: A total of 141 genes were screened out that exhibited different expression in all three chips. There were 74 upregulated and 67 downregulated genes in gene expression profiles of BE which were two times of that in CIM.CONCLUSION: There is a difference in gene expression level between BE and CIM epithelia. These 141 genes probably relate to the occurrence and development of BE and the progression to adenocarcinoma.

  18. Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

    Le Goff, Gaelle C; Desmet, Cloé; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-12-15

    We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures. PMID:20663657

  19. DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells

    Suresh V. Kuchipudi

    2015-06-01

    Full Text Available The data described in this article pertain to the article by Kuchipudi et al. (2014 titled “Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses” [1]. While infection of chickens with highly pathogenic avian influenza (HPAI H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEOdatabase with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014 [1].

  20. Gene expression profile analysis of genes in rat hippocampus from antidepressant treated rats using DNA microarray

    Shin Minkyu

    2010-11-01

    Full Text Available Abstract Background The molecular and biological mechanisms by which many antidepressants function are based on the monoamine depletion hypothesis. However, the entire cascade of mechanisms responsible for the therapeutic effect of antidepressants has not yet been elucidated. Results We used a genome-wide microarray system containing 30,000 clones to evaluate total RNA that had been isolated from the brains of treated rats to identify the genes involved in the therapeutic mechanisms of various antidepressants, a tricyclic antidepressant (imipramine. a selective serotonin reuptake inhibitor (fluoxetine, a monoamine oxidase inhibitor (phenelzine and psychoactive herbal extracts of Nelumbinis Semen (NS. To confirm the differential expression of the identified genes, we analyzed the amount of mRNA that was isolated from the hippocampus of rats that had been treated with antidepressants by real-time RT-PCR using primers specific for selected genes of interest. These data demonstrate that antidepressants interfere with the expression of a large array of genes involved in signaling, survival and protein metabolism, suggesting that the therapeutic effect of these antidepressants is very complex. Surprisingly, unlike other antidepressants, we found that the standardized herbal medicine, Nelumbinis Semen, is free of factors that can induce neurodegenerative diseases such as caspase 8, α-synuclein, and amyloid precursor protein. In addition, the production of the inflammatory cytokine, IFNγ, was significantly decreased in rat hippocampus in response to treatment with antidepressants, while the inhibitory cytokine, TGFβ, was significantly enhanced. Conclusions These results suggest that antidepressants function by regulating neurotransmission as well as suppressing immunoreactivity in the central nervous system.

  1. cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, Pinctada fucata martensii.

    Shi, Yaohua; Zheng, Xing; Zhan, Xin; Wang, Aimin; Gu, Zhifeng

    2016-06-01

    Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E ≤ -10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster. PMID:27184264

  2. DNA Micro-Array Gene Expression Profi ling of Angiogenesis in Collagen Gel Culture

    Masumi Akita; Keiko Fujita

    2008-01-01

    We examined angiogenesis-related gene expression profiles using collagen gel culture and a DNA chip. After isolation of total RNA from cultures before and after capillary tube formation, a mouse whole-genome array study was performed. Seventy-three out of over 35,000 transcripts were expressed after capillary tube formation. The majority of genes did not show any significant differences between before and after capillary tube formation. However, there were 7 upregulated genes; tumor necrosis ...

  3. Monitoring of malaria parasite resistance to chloroquine and sulphadoxine-pyrimethamine in the Solomon Islands by DNA microarray technology

    Felger Ingrid

    2010-10-01

    Full Text Available Abstract Background Little information is available on resistance to anti-malarial drugs in the Solomon Islands (SI. The analysis of single nucleotide polymorphisms (SNPs in drug resistance associated parasite genes is a potential alternative to classical time- and resource-consuming in vivo studies to monitor drug resistance. Mutations in pfmdr1 and pfcrt were shown to indicate chloroquine (CQ resistance, mutations in pfdhfr and pfdhps indicate sulphadoxine-pyrimethamine (SP resistance, and mutations in pfATPase6 indicate resistance to artemisinin derivatives. Methods The relationship between the rate of treatment failure among 25 symptomatic Plasmodium falciparum-infected patients presenting at the clinic and the pattern of resistance-associated SNPs in P. falciparum infecting 76 asymptomatic individuals from the surrounding population was investigated. The study was conducted in the SI in 2004. Patients presenting at a local clinic with microscopically confirmed P. falciparum malaria were recruited and treated with CQ+SP. Rates of treatment failure were estimated during a 28-day follow-up period. In parallel, a DNA microarray technology was used to analyse mutations associated with CQ, SP, and artemisinin derivative resistance among samples from the asymptomatic community. Mutation and haplotype frequencies were determined, as well as the multiplicity of infection. Results The in vivo study showed an efficacy of 88% for CQ+SP to treat P. falciparum infections. DNA microarray analyses indicated a low diversity in the parasite population with one major haplotype present in 98.7% of the cases. It was composed of fixed mutations at position 86 in pfmdr1, positions 72, 75, 76, 220, 326 and 356 in pfcrt, and positions 59 and 108 in pfdhfr. No mutation was observed in pfdhps or in pfATPase6. The mean multiplicity of infection was 1.39. Conclusion This work provides the first insight into drug resistance markers of P. falciparum in the SI. The

  4. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA - Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    Hatt, Lotte; Aagaard, Mads M; Graakjaer, Jesper;

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of...... the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA...... with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes....

  5. Development and evaluation of a DNA microarray assay for the simultaneous detection of nine harmful algal species in ship ballast and seaport waters

    Chen, Xianfeng; Zhou, Qianjin; Duan, Weijun; Zhou, Chengxu; Duan, Lijun; Zhang, Huili; Sun, Aili; Yan, Xiaojun; Chen, Jiong

    2016-01-01

    Rapid, high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae, which are responsible for algal blooms, such as red and green tides. In this study, we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously, namely Alexandrium tamarense, Gyrodinium instriatum, Heterosigma akashiwo, Karenia mikimotoi, Prorocentrum donghaiense, Prorocentrum minimum, Ulva compressa, Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection (LOD) of 0.5 ng of genomic DNA (orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether, 230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%, respectively, relative to conventional morphological methods. This indicated that this high-throughput, automatic, and specific method is well suited for the detection of algae in water samples.

  6. An assessment on DNA microarray and sequence-based methods for the characterization of methicillin-susceptible Staphylococcus aureus from Nigeria

    Adebayo Osagie Shittu

    2015-10-01

    Full Text Available Staphylococcus aureus is an important human pathogen causing nosocomial and community-acquired infections worldwide. In the characterization of this opportunistic pathogen, DNA microarray hybridization technique is used as an alternative to sequence based genotyping to obtain a comprehensive assessment on the virulence, resistance determinants, and population structure. The objective of this study was to characterize a defined collection of S. aureus isolates from Nigeria using the microarray technique, and to assess the extent that it correlates with sequence-based genotyping methods. The clonal diversity and genomic content of 52 methicillin-susceptible Staphylococcus aureus (MSSA were investigated by spa typing, MLST and DNA microarray hybridization. More than half (55.8% of these isolates were associated with clonal complexes typically associated with methicillin-resistant S. aureus (MRSA clones i.e. CC1, CC5, CC8, CC30 and CC45. Certain genes linked with virulence (hlgA and clfA and adherence (ebpS, fnbA, sspA, sspB and sspP were detected in all isolates. A number of genes or gene clusters were associated with distinct clonal types. The enterotoxin gene cluster (egc was linked with CC5, CC25, CC30, CC45 and CC121, enterotoxin H gene (seh with CC1, exfoliative toxin D gene (etd with CC25 and CC80, and the epidermal cell differentiation inhibitor B gene (edinB with CC25, CC80 and CC152. The excellent agreement between data from DNA microarray and MLST in the delineation of Nigerian MSSA isolates indicates that the microarray technique is a useful tool to provide information on antibiotic resistance, clonal diversity and virulence factors associated with infection and disease.

  7. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

    Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

    2016-05-15

    Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. PMID:26735874

  8. A novel time-course cDNA microarray analysis method identifies genes associated with the development of cisplatin resistance.

    Whiteside, Martin A; Chen, Dung-Tsa; Desmond, Renee A; Abdulkadir, Sarki A; Johanning, Gary L

    2004-01-22

    In recent years, most cDNA microarray studies of chemotherapeutic drug resistance have not considered the temporal pattern of gene expression. The objective of this study was to examine systematically changes in gene expression of NCI-H226 and NCI-H2170 lung cancer cells treated weekly with IC10 doses of cisplatin. NCI-H226 lung cancer cells were treated weekly with an IC10 dose of cisplatin. Candidate genes with a fold change of 2.0 or more were identified from this study. A second experiment was conducted by exposing NCI-H2170 cells to cisplatin doses that were increased in week 4 and decreased in week 5. Overall, 44 genes were differentially expressed in both the NCI-H226 and NCI-H2170 cell lines. In the NCI-H2170 cell line, 24 genes had a twofold gene expression change from weeks 3 to 4. Real-time PCR found a significant correlation of the gene expression changes for seven genes of interest. This small time-ordered series identified novel genes associated with cisplatin resistance. This kind of analysis should be viewed as a first step towards building gene-regulatory networks. PMID:14737109

  9. Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA microarrays.

    Fröls, Sabrina; Gordon, Paul M K; Panlilio, Mayi Arcellana; Schleper, Christa; Sensen, Christoph W

    2007-08-15

    The spindle-shaped Sulfolobus virus SSV1 was the first of a series of unusual and uniquely shaped viruses isolated from hyperthermophilic Archaea. Using whole-genome microarrays we show here that the circular 15.5 kb DNA genome of SSV1 exhibits a chronological regulation of its transcription upon UV irradiation, reminiscent to the life cycles of bacteriophages and eukaryotic viruses. The transcriptional cycle starts with a small UV-specific transcript and continues with early transcripts on both its flanks. The late transcripts appear after the onset of viral replication and are extended to their full lengths towards the end of the approximately 8.5 h cycle. While we detected only small differences in genome-wide analysis of the host Sulfolobus solfataricus comparing infected versus uninfected strains, we found a marked difference with respect to the strength and speed of the general UV response of the host. Models for the regulation of the virus cycle, and putative functions of genes in SSV1 are presented. PMID:17467765

  10. Identifying type 1 diabetes candidate genes by DNA microarray analysis of islet-specific CD4+ T cells

    Gregory J. Berry

    2015-09-01

    Full Text Available Type 1 diabetes (T1D is a T cell-mediated autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells and is fatal unless treated with insulin. During the last four decades, multiple insulin-dependent diabetes (Idd susceptibility/resistance loci that regulate T1D development have been identified in humans and non-obese diabetic (NOD mice, an established animal model for T1D. However, the exact mechanisms by which these loci confer diabetes risk and the identity of the causative genes remain largely elusive. To identify genes and molecular mechanisms that control the function of diabetogenic T cells, we conducted DNA microarray analysis in islet-specific CD4+ T cells from BDC2.5 TCR transgenic NOD mice that contain the Idd9 locus from T1D-susceptible NOD mice or T1D-resistant C57BL/10 mice. Here we describe in detail the contents and analyses for these gene expression data associated with our previous study [1]. Gene expression data are available at the Gene Expression Omnibus (GEO repository from the National Center for Biotechnology Information (accession number GSE64674.

  11. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors.

    Weingeist, David M; Ge, Jing; Wood, David K; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A; Yaffe, Michael B; Floyd, Scott; Engelward, Bevin P

    2013-03-15

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects. PMID:23422001

  12. Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels

    Barat-Houari, M; Hilliou, F.; Jousset, F-X; Sofer, L; Deleury, E.; Rocher, J.; Ravallec, M.; Galibert, L; Delobel, P; Feyereisen, R.; Fournier, P.; Volkoff, A-N.

    2006-01-01

    Background Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as p...

  13. Phytochip: Development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species

    Noyer, Charlotte; Abot, Anne; Trouilh, Lidwine; Leberre, Veronique Anton; Dreanno, Catherine

    2015-01-01

    Detection of harmful algal blooms has become a challenging concern because of to the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip...

  14. Cross-species hybridizations on a multi-species cDNA microarray to identify evolutionarily conserved genes expressed in oocytes

    Palin Marie-France; Méthot Steve; Robert Claude; Vallée Maud; Sirard Marc-André

    2006-01-01

    Abstract Background Comparative genomic analysis using cDNA microarray is a new approach and a useful tool to identify important genetic sequences or genes that are conserved throughout evolution. Identification of these conserved sequences will help elucidate important molecular mechanisms or pathways common to many species. For example, the stockpiled transcripts in the oocyte necessary for successful fertilization and early embryonic development still remain relatively unknown. The objecti...

  15. Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of Listeria monocytogenes from Genomic DNA

    Borucki, Monica K.; Reynolds, James; Douglas R Call; Ward, Todd J.; Page, Brent; Kadushin, James

    2005-01-01

    Listeria monocytogenes is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar m...

  16. Functional demonstration of adaptive immunity in zebrafish using DNA vaccination

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja; Rasmussen, Jesper Skou; Kjær, Torben Egil; Vesely, Thomas

    Due to the well characterized genome, overall highly synteny with the human genome and its suitability for functional genomics studies, the zebrafish is considered to be an ideal animal model for basic studies of mechanisms of diseases and immunity in vertebrates including humans. While several...... studies have documented existence of a classical innate immune response, there is mainly indirect evidence of functional adaptive immunity. To address this aspect, groups of zebrafish were vaccinated with DNA-vaccines against the rhabdoviruses VHSV, IHNV and SVCV. Seven weeks later, the fish were...... challenged with SVCV by immersion. Despite some variability between replicate aquaria, there was a protective effect of the homologous vaccine and no effect of the heterologous vaccines. The results therefore confirm the existence of not only a well developed but also a fully functional adaptive immune...

  17. p53 Mutation analysis in breast tumors by a DNA microarray method.

    Tennis, Meredith; Krishnan, Shiva; Bonner, Matthew; Ambrosone, Christine B; Vena, John E; Moysich, Kirsten; Swede, Helen; McCann, Susan; Hall, Per; Shields, Peter G; Freudenheim, Jo L

    2006-01-01

    The p53 gene acts as a regulator of cell growth and DNA repair in normal cells; inactivation of the gene seems to lead to cancer. It is the most commonly mutated gene in human cancers, and a high-throughput sequencing method is needed for cancer etiology studies using large sample sets. In our population-based case-control study of breast cancer, the p53 gene was amplified by PCR for 392 subjects from seven hospitals in Western New York using the Affymetrix GeneChip technology. One hundred thirty-eight (35%) of the breast tumors had p53 mutations, of which 88% were located in exons 5 to 8. New hotspots were identified at codons 179, 195, 196, 213, 217, 249, 254, 278, 281, and 298, and previously reported hotspots were found at codons 175, 248, and 273. Manual sequencing for exons 5 to 9 of the p53 gene was done for 139 tumors to validate the Affymetrix assay. The two methods had 100% concordance for mutations detectable by the Affymetrix assay. We also successfully assayed paraffin-embedded breast and lung tumors from as early as 1958 and employed a nested PCR strategy to improve weak PCR amplification. To have statistical power, the investigation of gene environment interactions and cancer requires a large number of tumor analyses, which are frequently only available from archived tissue from multiple sources. We have shown the utility of the Affymetrix GeneChip method under these challenging conditions and provided new data for the mutational spectra of breast cancer in a population-based study. PMID:16434591

  18. Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis

    Indranil Chattopadhyay; Sujala Kapur; Joydeep Purkayastha; Rupkumar Phukan; Amal Kataki; Jagadish Mahanta; Sunita Saxena

    2007-01-01

    AIM: To identify alterations in genes and molecular functional pathways in esophageal cancer in a high incidence region of India where there is a widespread use of tobacco and betel quid with fermented areca nuts.METHODS: Total RNA was isolated from tumor and matched normal tissue of 16 patients with esophageal squamous cell carcinoma. Pooled tumor tissue RNA was labeled with Cy3-dUTP and pooled normal tissue RNA was labeled with Cy5-dUTP by direct labeling method.The labeled probes were hybridized with human 10K cDNA chip and expression profiles were analyzed by Genespring GX V 7.3 (Silicon Genetics).RESULTS: Nine hundred twenty three genes were differentially expressed. Of these, 611 genes were upregulated and 312 genes were downregulated. Using stringent criteria (P ≤ 0.05 and ≥ 1.5 fold change),127 differentially expressed genes (87 upregulated and 40 downregulated) were identified in tumor tissue. On the basis of Gene Ontology, four different molecular functional pathways (MAPK pathway,G-protein coupled receptor family, ion transport activity,and serine or threonine kinase activity) were most significantly upregulated and six different molecular functional pathways (structural constituent of ribosome,endopeptidase inhibitor activity, structural constituent of cytoskeleton, antioxidant activity, acyl group transferase activity, eukaryotic translation elongation factor activity)were most significantly downregulated.CONCLUSION: Several genes that showed alterations in our study have also been reported from a high incidence area of esophageal cancer in China. This indicates that molecular profiles of esophageal cancer in these two different geographic locations are highly consistent.

  19. Research on DNA microarray chip method for detecting drug resistance of Mycobacterium tuberculosis%DNA 微阵列芯片法用于检测结核分枝杆菌耐药性的研究

    刘亚芹; 杨振斌; 冯冬霞; 王海英

    2014-01-01

    目的:通过与传统的比例法在结核分枝杆菌耐药性检测的比较,评价 DNA 微阵列法用于检测结核分枝杆菌耐药性的可行性。方法随机抽取本院2012年1月至2013年3月从临床标本中分离培养所得的结核分枝杆菌54株,通过 DNA 微阵列法和比例法分别进行异烟肼和利福平的耐药性检测并对结果进行比较分析。结果以比例法为金标准,DNA 微阵列法对异烟肼和利福平的耐药检测结果与比例法的符合率分别为75.0%、91.0%。结论 DNA 微阵列技术适用于临床一线耐药结核分枝杆菌的快速筛查。%Objective To evaluate the feasibility of the DNA microarray method used in detecting the drug resistance of Myco-bacterium tuberculosis by comparing the traditional proportion method and the DNA microarray method for detecting the drug re-sistance of Mycobacterium tuberculosis.Methods 54 strains of Mycobacterium tuberculosis isolated from clinical specimens in our hospital from January 2012 to March 2013 were randomly extracted and their resistance to isonicotinic acid hydrazide (INH)and rifampicin(RFP)was detected by the DNA microarray method and the proportion method.The detection results were performed the comparative analysis.Results With the proportion method as the golden standard,the coincidence rates of the DNA microarray method for detecting the Mycobacterium tuberculosis resistance to INH and RFP were 75% and 91.0% respectively.Conclusion The DNA microarray technique is suitable for the rapid screening of clinical first-line drug resistant Mycobacterium tuberculosis.

  20. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

    Paniego Norma

    2008-01-01

    Full Text Available Abstract Background Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion

  1. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparisons with Other Methods

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site (Hanford Reach of the Columbia River (HRCR), 11 strains), Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  2. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  3. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  4. Carbohydrate microarrays

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

    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-based technol......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......-based technology has been widely employed for rapid analysis of the glycan binding properties of lectins and antibodies, the quantitative measurements of glycan-protein interactions, detection of cells and pathogens, identification of disease-related anti-glycan antibodies for diagnosis, and fast assessment of...

  5. 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

    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

  6. Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

    Walsh, Fiona

    2010-06-01

    A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

  7. Estrogenic activity of bio-degradation products of C-heavy oil revealed by gene-expression profiling using an oligo-DNA microarray system

    Degradation of heavy oil by bacteria to decompose organic compounds such as aliphatic and aromatic hydrocarbons has been used in bioremediation. However, the biological and environmental effects of the degradation products including intermediates are still not clear. Here, we monitored the degradation of C-heavy oil by analyzing the products formed in cultures with oil-degrading bacteria (complex microbes or a single bacterial strain). Furthermore, proliferation assays using breast cancer MCF-7 cells and gene-expression profiling of MCF-7 cells using oligonucleotide-DNA microarrays were performed to evaluate the estrogenic activity of the degradation products. While the products did not show any significant cell-proliferative activity, the oil samples cultured for longer periods (2–3 months), whether cultured with mixed microbes or a single bacterial strain, showed gene-expression profiles similar to that of 17β-estradiol (E2). These results suggest that oil-degradation products have estrogenic activity, and estrogen-like components could possibly be produced during the degradation process. - Highlights: ► Oligonucleotide-DNA microarrays were used to monitor bio-degradation of C-heavy oil. ► The oil samples showed gene-expression profiles similar to that of 17β-estradiol. ► Estrogen-like components were produced during the degradation process. ► The effect of bio-degradation products on ecosystems needs more attention. ► DNA microarray assays may be useful for monitoring such products. - We found that the degradation products of C-heavy oil contained estrogen-like components, suggesting the effect of bio-degradation products on ecosystems.

  8. Aptamer Microarrays

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  9. Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    Rollinson David

    2008-12-01

    Full Text Available Abstract Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs libraries, suppression subtractive hybridization (SSH libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7

  10. Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray

    Coser, Kathryn R.; Chesnes, Jessica; Hur, Jingyung; Ray, Sandip; Isselbacher, Kurt J.; Shioda, Toshi

    2003-01-01

    To obtain comprehensive information on 17β-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0–100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growt...

  11. Modulation of GdCl3 and Angelica Sinensis polysaccharides on differentially expressed genes in liver of hepatic immunological injury mice by cDNA microarray

    Hong Ding; Gang-Gang Shi; Xin Yu; Jie-Ping Yu; Jie-An Huang

    2003-01-01

    AIM: To study the modulating effect of GdCl3 and Angelica Sinensis polysaccharides (ASP) on differentially expressed genes in liver of hepatic immunological mice by cDNA microarray.METHODS: Hepatic immunological injury was induced by lipopolysaccharide (LPS ip, 0.2 mg.kg-1) in bacillus calmetteguerin (BCG ip, 1 mg.kg-1) primed mice; A single dose of 20 mg.kg-1 GdCl3 was simultaneously pretreated and 30 mg.kg-1 ASP (ig, qd×7 d) was administrated when the BCG+LPS was primed. The mice were sacrificed at the end of the 7th day after ip LPS for 6 h and the liver was removed quickly. The PCR products of 512 genes were spotted onto a chemical material-coated glass plate in array. The DNAs were fixed to the glass plate after series of treatments. The total RNAs were isolated from the liver tissue, and were purified to mRNAs by Oligotex.Both mRNAs from the normal liver tissue and the liver tissue from the mice with hepatic immunological injury or that pretreated with GdCl3 or ASP were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After highstringent washing, the cDNA microarray was scanned for fluorescent signals and showed differences between the two tissues.RESULTS: Among the 512 target genes, 18 differed in liver tissue of hepatic immunological injury mice, and 6 differed in those pretreated by ASP, 7 differed in those pretreated by GdCl3.CONCLUSION: cDNA microarray technique is effective in screening the differentially expressed genes between two different kinds of tissue. Further analysis of those obtained genes will be helpful to understand the molecular mechanism of hepatic immunological injury and to study the intervention of drug. Both ASP and GdCl3 can decrease the number of the differentially expressed genes in liver tissue of mice with hepatic immunological injury.

  12.  DNA microarray-based gene expression profiling in diagnosis, assessing prognosis and predicting response to therapy in colorectal cancer

    Przemysław Kwiatkowski

    2012-06-01

    Full Text Available  Colorectal cancer is the most common cancer of the gastrointestinal tract. It is considered as a biological model of a certain type of cancerogenesis process in which progression from an early to late stage adenoma and cancer is accompanied by distinct genetic alterations.Clinical and pathological parameters commonly used in clinical practice are often insufficient to determine groups of patients suitable for personalized treatment. Moreover, reliable molecular markers with high prognostic value have not yet been determined. Molecular studies using DNA-based microarrays have identified numerous genes involved in cell proliferation and differentiation during the process of cancerogenesis. Assessment of the genetic profile of colorectal cancer using the microarray technique might be a useful tool in determining the groups of patients with different clinical outcomes who would benefit from additional personalized treatment.The main objective of this study was to present the current state of knowledge on the practical application of gene profiling techniques using microarrays for determining diagnosis, prognosis and response to treatment in colorectal cancer.

  13. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

    Lotte Hatt

    Full Text Available Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

  14. Development of the first marmoset-specific DNA microarray (EUMAMA: a new genetic tool for large-scale expression profiling in a non-human primate

    Waegele Brigitte

    2007-06-01

    Full Text Available Abstract Background The common marmoset monkey (Callithrix jacchus, a small non-endangered New World primate native to eastern Brazil, is becoming increasingly used as a non-human primate model in biomedical research, drug development and safety assessment. In contrast to the growing interest for the marmoset as an animal model, the molecular tools for genetic analysis are extremely limited. Results Here we report the development of the first marmoset-specific oligonucleotide microarray (EUMAMA containing probe sets targeting 1541 different marmoset transcripts expressed in hippocampus. These 1541 transcripts represent a wide variety of different functional gene classes. Hybridisation of the marmoset microarray with labelled RNA from hippocampus, cortex and a panel of 7 different peripheral tissues resulted in high detection rates of 85% in the neuronal tissues and on average 70% in the non-neuronal tissues. The expression profiles of the 2 neuronal tissues, hippocampus and cortex, were highly similar, as indicated by a correlation coefficient of 0.96. Several transcripts with a tissue-specific pattern of expression were identified. Besides the marmoset microarray we have generated 3215 ESTs derived from marmoset hippocampus, which have been annotated and submitted to GenBank [GenBank: EF214838 – EF215447, EH380242 – EH382846]. Conclusion We have generated the first marmoset-specific DNA microarray and demonstrated its use to characterise large-scale gene expression profiles of hippocampus but also of other neuronal and non-neuronal tissues. In addition, we have generated a large collection of ESTs of marmoset origin, which are now available in the public domain. These new tools will facilitate molecular genetic research into this non-human primate animal model.

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

    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

  16. Mitochondrial DNA content contributes to climate adaptation using Chinese populations as a model.

    Yao-Ting Cheng

    Full Text Available Maintaining a balance between ATP synthesis and heat generation is crucial for adapting to changes in climate. Variation in the mitochondrial DNA (mtDNA, which encodes 13 subunits of the respiratory chain complexes, may contribute to climate adaptation by regulating thermogenesis and the use of bioenergy. However, studies looking for a relationship between mtDNA haplogroups and climate have obtained mixed results, leaving unresolved the role of mtDNA in climate adaptation. Since mtDNA content can regulate human bioenergy processes and is known to influence many physiological traits and diseases, it is possible that mtDNA content contributes to climate adaptation in human populations. Here, we analyze the distribution of mtDNA content among 27 Chinese ethnic populations residing across China and find a significant association between mtDNA content and climate, with northern populations having significantly higher mtDNA content than southern populations. Functional studies have shown that high mtDNA content correlates with an increase in the expression of energy metabolism enzymes, which may accelerate thermogenesis. This suggests that the significantly higher mtDNA content observed in northern populations may confer a selective advantage in adapting to colder northern climates.

  17. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong;

    2011-01-01

    article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of......Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this...... RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal...

  18. Radioactive human cDNA microarray: comparison of anti-cancer effect between 8-chloro-cAMP and 8-chloro-adenosine

    Eight-chloroadenosine 3.5-monophosphate (8-Cl-cAMP), a site-selective cyclic adenosine 3,5-monophosphate (cAMP) analogue, exhibits growth inhibition in a broad spectrum of cancer cell lines. Nonetheless, there has been a debate as to whether it is a prodrug for its 8-Cl-adenosine metabolite. Using human cDNA microarray, we investigated a pattern of gene regulation under the treatment of 8-Cl-cAMP and 8-Cl-adenosine in PC3M (ATCC CRL-1435; human prostate cancer cell line). The general methodology of arraying is based on the procedures of DeRisi, et al. (Nature Genetics 14: 48, 1996). The gene expression in PC3M by treating 8-Cl-cAMP 8-Cl-adenosine demonstrated a different pattern compared with nontreated control: bifurcated types for up-regulation but a single-directed type for down-regulation. In summary, we demonstrated that the human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of anti-cancer therapeutic agents, such as 8-Cl-cAMP vs. 8-Cl-adenosine, which has been controverted for a long time

  19. Microarray results: how accurate are they?

    Mane Shrikant

    2002-08-01

    Full Text Available Abstract Background DNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially. Results In this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene. Conclusions In view of these pitfalls, data from microarray analysis need to be interpreted cautiously.

  20. DNA Microarray Based on Arrayed-Primer Extension Technique for Identification of Pathogenic Fungi Responsible for Invasive and Superficial Mycoses▿

    Campa, Daniele; Tavanti, Arianna; Gemignani, Federica; Mogavero, Crocifissa S.; Bellini, Ilaria; Bottari, Fabio; Barale, Roberto; Landi, Stefano; Senesi, Sonia

    2007-01-01

    An oligonucleotide microarray based on the arrayed-primer extension (APEX) technique has been developed to simultaneously identify pathogenic fungi frequently isolated from invasive and superficial infections. Species-specific oligonucleotide probes complementary to the internal transcribed spacer 1 and 2 (ITS1 and ITS2) region were designed for 24 species belonging to 10 genera, including Candida species (Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida tropi...

  1. The phytoplankton chip - development and assessment of a DNA microarray as a reliable tool for monitoring of phytoplankton

    Gescher, Christine

    2007-01-01

    One microarray, the Phytoplankton Chip was developed . Phytoplankton field samples were taken at the island of Helgoland in the North Sea from 2004 to 2006 at regular intervals. For the phytoplankton community, only the > 20 mikrometer size fraction is identified on a daily basis. For picoplanktonic groups, light microscopy can not differentiate taxa or species. The phyto- and especially picoplanktonic dynamics were successfully analyzed with the Phytoplankton Chip in these three annual cycle...

  2. AN INTELLIGENT SEGMENTATION ALGORITHM FOR MICROARRAY IMAGE PROCESSING

    P.Rajkumar

    2013-06-01

    Full Text Available Microarray technology consists of an array of thousands of microscopic spots of DNA oligonucleotides attached to a solid surface. It is a very powerful technique for analyzing gene expressions as well as to explore the underlying genetic causes of many human diseases. There are numerous applications of this technology, including environmental health research, drug design and discovery, clinical diagnosis and treatment and in cancer detection. The spots, which represent genes in microarray experiment contains the quantitative information that needs to be extracted accurately. For this process, preprocessing of microarray plays an essential role and it is also influential in future steps of the analysis. The three microarray preprocessing steps include gridding, segmentation and quantification. The first step is gridding, refers to the identification of the centre coordinates of each spot. The second step is segmentation, refers to the process of separating foreground and background fluorescence intensities. Segmentation is very important step as it directly affects the accuracy of gene expression analysis in the data mining process that follows. Accurate segmentation is one of the vital steps in microarray image processing. A novel method for segmentation of microarray image is proposed which accurately segment the spots from background when compared with adaptive threshold, combined global and local thresholdand fuzzy c-means clustering methods. Experimental results show that our proposed method provides better segmentation and improved intensity values than the above existing methods.

  3. Microarray analysis in the archaeon Halobacterium salinarum strain R1.

    Jens Twellmeyer

    Full Text Available BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

  4. The elusive nature of adaptive mitochondrial DNA evolution of an Arctic lineage prone to frequent introgression

    Melo-Ferreira, Jose; Vilela, Joana; Fonseca, Miguel M.;

    2014-01-01

    Mitochondria play a fundamental role in cellular metabolism, being responsible for most of the energy production of the cell in the oxidative phosphorylation (OXPHOS) pathway. Mitochondrial DNA (mtDNA) encodes for key components of this process, but its direct role in adaptation remains far from...... understood. Hares (Lepus spp.) are privileged models to study the impact of natural selection on mitogenomic evolution because 1) species are adapted to contrasting environments, including arctic, with different metabolic pressures, and 2) mtDNA introgression from arctic into temperate species is widespread...... lie on complex interactions with nuclear encoded peptides. Also, a cloverleaf structure was detected in the control region only from the arctic mtDNA lineage, which may influence mtDNA replication and transcription. These results suggest that adaptation impacted the evolution of hare mtDNA and may...

  5. Gene expression in Florida red tide dinoflagellate Karenia brevis: analysis of an expressed sequence tag library and development of DNA microarray.

    Lidie, Kristy B; Ryan, James C; Barbier, Michele; Van Dolah, Frances M

    2005-01-01

    Karenia brevis (Davis) is the dinoflagellate responsible for nearly annual red tides in the Gulf of Mexico. Although the mechanisms regulating the growth and toxicity of this problematic organism are of considerable interest, little information is available on its molecular biology. We therefore constructed a complementary DNA library from which to gain insight into its expressed genome and to develop tools for studying its gene expression. Large-scale sequencing yielded 7001 high-quality expressed sequence tags (ESTs), which clustered into 5280 unique gene groups. The vast majority of genes expressed fell into a low-abundance class, with the highest expressed gene accounting for only 1% of the total ESTs. Approximately 29% of genes were found to have similarity to known sequences in other organisms after BLAST similarity comparisons to the GenBank public protein database using a cutoff of P < 10e(-4). We identified for the first time in a dinoflagellate a suite of conserved eukaryotic genes involved in cell cycle control, intracellular signaling, and the transcription and translation machinery. At least 40% of gene clusters displayed single nucleotide polymorphisms, suggesting the presence of multiple gene copies. The average GC content of ESTs was 51%, with a slight preference for G or C in the third codon position (53.5%). The ESTs were used to develop an oligonucleotide microarray containing 4629 unique features and 3462 replicate probes. Microarray labeling has been optimized, and the microarray has been validated for probe specificity and reproducibility. This is the first information to be developed on the expressed genome of K. brevis and provides the basis from which to begin functional genomic studies on this harmful algal bloom species. PMID:15976935

  6. Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.

    Fan, Qing-Jie; Yan, Feng-Xia; Qiao, Guang; Zhang, Bing-Xue; Wen, Xiao-Peng

    2014-01-01

    Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in

  7. Fast DNA serotyping and antimicrobial resistance gene determination of salmonella enterica with an oligonucleotide microarray-based assay.

    Braun, Sascha D; Ziegler, Albrecht; Methner, Ulrich; Slickers, Peter; Keiling, Silke; Monecke, Stefan; Ehricht, Ralf

    2012-01-01

    Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4-5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping. PMID:23056321

  8. Identification of late O{sub 3}-responsive genes in Arabidopsis thaliana by cDNA microarray analysis

    D' Haese, D. [Univ. of Antwerp, Dept. of Biology, Antwerp (BE) and Univ. of Newcastle, School of Biology and Psychology, Div. of Biology, Newcastle-Upon-Tyne (United Kingdom); Horemans, N.; Coen, W. De; Guisez, Y. [Univ. of Antwerp, Dept. of Biology, Antwerp (Belgium)

    2006-09-15

    To better understand the response of a plant to 0{sub 3} stress, an integrated microarray analysis was performed on Arabidopsis plants exposed during 2 days to purified air or 150 nl l{sup -1} O{sub 3}, 8 h day-l. Agilent Arabidopsis 2 Oligo Microarrays were used of which the reliability was confirmed by quantitative real-time PCR of nine randomly selected genes. We confirmed the O{sub 3} responsiveness of heat shock proteins (HSPs), glutathione-S-tranferases and genes involved in cell wall stiffening and microbial defence. Whereas, a previous study revealed that during an early stage of the O{sub 3} stress response, gene expression was strongly dependent on jasmonic acid and ethylene, we report that at a later stage (48 h) synthesis of jasrnonic acid and ethylene was downregulated. In addition, we observed the simultaneous induction of salicylic acid synthesis and genes involved in programmed cell death and senescence. Also typically, the later stage of the response to O{sub 3} appeared to be the induction of the complete pathway leading to the biosynthesis of anthocyanin diglucosides and the induction of thioredoxin-based redox control. Surprisingly absent in the list of induced genes were genes involved in ASC-dependent antioxidation, few of which were found to be induced after 12 h of 0{sub 3} exposure in another study. We discuss these and other particular results of the microarray analysis and provide a map depicting significantly affected genes and their pathways highlighting their interrelationships and subcellular localization. (au)

  9. Studying of the DNA DSBs repair in the adaptive response of human lymphocytes

    Human lymphocytes exposed to very low doses of DNA damaging agents may become less sensitive to subsequent higher doses of the DNA damaging agent. This phenomenon is called the adaptive response. The aim of this project was to study the significance of DNA double-strand break (DSBs) repair in the adaptive response of human lymphocytes exposed to ionizing radiation. Human lymphocytes isolated from whole blood and stimulated with hemagglutynin (PHA) were irradiated with adaptive dose (5 cGy of X-rays) and then challenge dose of 2 Gy in case of micronuclei assay and 10 Gy in case of comet assay. The frequency of micronuclei in adapted lymphocytes was about 30% lower than expected for an additive effect of both, adaptive and challenge doses, applied separately. Estimation of DSBs was carried out with the use of single cell gel electrophoresis assay (comet assay) in neutral pH and to complement these results with pulse-field gel electrophoresis (PFGE). The use of both PFGE and comet assay allowed us to suggest that lower damage revealed in the adapted lymphocytes at the chromosomal level was unrelated to initial level of DSBs in DNA. The differences between kinetics of DNA repair in the adapted and non-adapted lymphocytes were not significant. (author)

  10. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

    Maija Wolf

    2004-05-01

    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  11. Resolution of large and small differences in gene expression using models for the Bayesian analysis of gene expression levels and spotted DNA microarrays

    Townsend Jeffrey P

    2004-05-01

    Full Text Available Abstract Background The detection of small yet statistically significant differences in gene expression in spotted DNA microarray studies is an ongoing challenge. Meeting this challenge requires careful examination of the performance of a range of statistical models, as well as empirical examination of the effect of replication on the power to resolve these differences. Results New models are derived and software is developed for the analysis of microarray ratio data. These models incorporate multiplicative small error terms, and error standard deviations that are proportional to expression level. The fastest and most powerful method incorporates additive small error terms and error standard deviations proportional to expression level. Data from four studies are profiled for the degree to which they reveal statistically significant differences in gene expression. The gene expression level at which there is an empirical 50% probability of a significant call is presented as a summary statistic for the power to detect small differences in gene expression. Conclusions Understanding the resolution of difference in gene expression that is detectable as significant is a vital component of experimental design and evaluation. These small differences in gene expression level are readily detected with a Bayesian analysis of gene expression level that has additive error terms and constrains samples to have a common error coefficient of variation. The power to detect small differences in a study may then be determined by logistic regression.

  12. Gene expression analysis of radiation-induced apoptosis in human squamous cell carcinoma with different p53 status using DNA microarray

    Yane, Katsunari; Ota, Ichiro; Yuki, Kazue; Hosoi, Hiroshi; Takahashi, Akihisa; Ohnishi, Ken; Ohnishi, Takeo [Nara Medical Univ., Kashihara (Japan)

    2002-06-01

    To identify genes that are associated with radiosensitivity in different p53 gene status, we used a cDNA microarray containing 161 genes related to apoptosis. Two kind of cells of wild-type p53 cells (SAS/neo) and mutation-type p53 cells (SAS/mp53) were utilized to clarify the expression of genes which induce apoptosis at different times after 6 Gy irradiation. The gene expression of casper was over 10 fold high in gene expression at 36 hr in SAS/neo cells as compared as unirradiated cells, whereas the gene expression of caspase disappeared at 2 hr in SAS/mp53 cells. These results suggest that the casper gene might play an important role for radiosensitivity. (author)

  13. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  14. Protein Microarray On-Demand: A Novel Protein Microarray System

    Chatterjee, Deb K.; Sitaraman, Kalavathy; Baptista, Cassio; Hartley, James; Hill, Thomas M.; David J. Munroe

    2008-01-01

    We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity bin...

  15. Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray.

    Coser, Kathryn R; Chesnes, Jessica; Hur, Jingyung; Ray, Sandip; Isselbacher, Kurt J; Shioda, Toshi

    2003-11-25

    To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed approximately 3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced approximately 1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor alpha and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions. PMID:14610279

  16. Recent advances of protein microarrays

    Hultschig, Claus; Kreutzberger, Jürgen; Seitz, Harald; Konthur, Zoltán; Büssow, Konrad; Lehrach, Hans

    2006-01-01

    Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss ...

  17. Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses

    On, Stephen L.W.; Dorrell, N.; Petersen, L.;

    2006-01-01

    Molecular epidemiological studies of the enteric pathogen Campylobacter jejuni have suggested that not all animal isolates are equally pathogenic to humans. We examined the use of numerical analysis of whole-genomotype data as a potential tool for evaluating C. jejuni virulence potential. Whole......-genome microarray analysis was used to determine the gene-level complementarity of 12 Danish strains to the pathogenic, genome-sequenced strain NCTC 11168. Cytolethal distending toxin (CDT) and haemolysin activities, and survival characteristics under aerobic conditions at room temperature were also determined....... Among the strains examined, 439 genes were polymorphic. Numerical analysis of these data by use of the squared Euclidean distance coefficient and Ward's clustering method clearly delineated strains into two clusters. CDT and haemolysin activities of cluster 1 strains were not statistically significantly...

  18. Non-coding DNA programs express adaptation and its universal law

    Azbel', Mark Ya.

    2007-01-01

    Significant fraction (98.5% in humans) of most animal genomes is non- coding dark matter. Its largely unknown function (1-5) is related to programming (rather than to spontaneous mutations) of accurate adaptation to rapidly changing environment. Programmed adaptation to the same universal law for non-competing animals from anaerobic yeast to human is revealed in the study of their extensively quantified mortality (6-21). Adaptation of animals with removed non-coding DNA fractions may specify ...

  19. Zinc finger recombinases with adaptable DNA sequence specificity.

    Chris Proudfoot

    Full Text Available Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural 'pseudosites' are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene, mediated by zinc finger recombinases (ZFRs, chimaeric enzymes with linked zinc finger (DNA recognition and recombinase (catalytic domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.

  20. Phytochip: development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species.

    Noyer, Charlotte; Abot, Anne; Trouilh, Lidwine; Leberre, Véronique Anton; Dreanno, Catherine

    2015-05-01

    Detection of harmful algal blooms has become a challenging concern because of the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip) for the simultaneous detection of multiple HAB species with a particular emphasis on Pseudo-nitzschia species. Oligonucleotide probes were designed along the rRNA operon. After DNA extraction, the target rDNA genes were amplified and labeled using an asymmetric PCR; then, the amplicons were hybridized to the oligonucleotide probes present on the chips. The total assay from seawater sampling to data acquisition can be performed within a working day. Specificity and sensitivity were assessed by using monoclonal cultures, mixtures of species and field samples spiked with a known amount of cultured cells. The Phytochip with its 81 validated oligonucleotide probes was able to detect 12 species of Pseudo-nitzschia and 11 species of dinoflagellates among which were 3 species of Karenia and 3 species of Alexandrium. The Phytochip was applied to environmental samples already characterized by light microscopy and cloned into DNA libraries. The hybridizations on the Phytochip were in good agreement with the sequences retrieved from the clone libraries and the microscopic observations. The Phytochip enables a reliable multiplex detection of phytoplankton and can assist a water quality monitoring program as well as more general ecological research. PMID:25765159

  1. Combining Affymetrix microarray results

    Doerge RW

    2005-03-01

    Full Text Available Abstract Background As the use of microarray technology becomes more prevalent it is not unusual to find several laboratories employing the same microarray technology to identify genes related to the same condition in the same species. Although the experimental specifics are similar, typically a different list of statistically significant genes result from each data analysis. Results We propose a statistically-based meta-analytic approach to microarray analysis for the purpose of systematically combining results from the different laboratories. This approach provides a more precise view of genes that are significantly related to the condition of interest while simultaneously allowing for differences between laboratories. Of particular interest is the widely used Affymetrix oligonucleotide array, the results of which are naturally suited to a meta-analysis. A simulation model based on the Affymetrix platform is developed to examine the adaptive nature of the meta-analytic approach and to illustrate the usefulness of such an approach in combining microarray results across laboratories. The approach is then applied to real data involving a mouse model for multiple sclerosis. Conclusion The quantitative estimates from the meta-analysis model tend to be closer to the "true" degree of differential expression than any single lab. Meta-analytic methods can systematically combine Affymetrix results from different laboratories to gain a clearer understanding of genes' relationships to specific conditions of interest.

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

    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/

  3. Dysregulated Mitochondrial Genes and Networks with Drug Targets in Postmortem Brain of Patients with Posttraumatic Stress Disorder (PTSD Revealed by Human Mitochondria-Focused cDNA Microarrays

    Yan A. Su, Jun Wu, Lei Zhang, Qiuyang Zhang, David M. Su, Ping He, Bi-Dar Wang, He Li, Maree J. Webster, Traumatic Stress Brain Study Group, Owen M. Rennert, Robert J. Ursano

    2008-01-01

    Full Text Available Posttraumatic stress disorder (PTSD is associated with decreased activity in the dorsolateral prefrontal cortex (DLPFC, the brain region that regulates working memory and preparation and selection of fear responses. We investigated gene expression profiles in DLPFC Brodmann area (BA 46 of postmortem patients with (n=6 and without PTSD (n=6 using human mitochondria-focused cDNA microarrays. Our study revealed PTSD-specific expression fingerprints of 800 informative mitochondria-focused genes across all of these 12 BA46 samples, and 119 (±>1.25, p<0.05 and 42 (±>1.60, p<0.05 dysregulated genes between the PTSD and control samples. Quantitative RT-PCR validated the microarray results. These fingerprints can essentially distinguish the PTSD DLPFC BA46 brains from controls. Of the 119 dysregulated genes (±≥125%, p<0.05, the highest percentages were associated with mitochondrial dysfunction (4.8%, p=6.61x10-6, oxidative phosphorylation (3.8%, p=9.04x10-4, cell survival-apoptosis (25.2%, p<0.05 and neurological diseases (23.5%, p<0.05. Fifty (50 dysregulated genes were present in the molecular networks that are known to be involved in neuronal function-survival and contain 7 targets for neuropsychiatric drugs. Thirty (30 of the dysregulated genes are associated with a number of neuropsychiatric disorders. Our results indicate mitochondrial dysfunction in the PTSD DLPFC BA46 and provide the expression fingerprints that may ultimately serve as biomarkers for PTSD diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of PTSD.

  4. Chromosome Microarray.

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  5. Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

    Telma Quintela

    Full Text Available The choroid plexus (CP are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF, the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

  6. Direct calibration of PICKY-designed microarrays

    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.

  7. Application of high-density DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.

    Jessica Vanhomwegen

    Full Text Available BACKGROUND: Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we designed and evaluated the performances of a resequencing microarray (RMA, the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components. This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays. CONCLUSIONS/SIGNIFICANCE: The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.

  8. Molecular mechanism of radioadaptive response: A cross-adaptive response for enhanced repair of DNA damage in adapted cells

    The radioadaptive response (RAR) has been attributed to the induction of a repair mechanism by low doses of ionizing radiation, but the molecular nature of the mechanism is not yet elucidated. We have characterized RAR in a series of experiments in cultured Chinese hamster V79 cells. A 4-h interval is required for the full expression of RAR, which decays with the progression of cell proliferation. Treatments with inhibitors of poly(ADP-ribose) polymerase, protein- or RNA synthesis, and protein kinase C suppress the RAR expression. The RAR cross-reacts on clastogenic lesions induced by other physical and chemical DNA-damaging agents. The presence of newly synthesised proteins has been detected during the expression period. Experiments performed using single-cell gel electrophoresis provided more direct evidence for a faster and enhaced DNA repair rate in adapted cells. Here, using single-cell gel electrophoresis, a cross-adaptive response has been demonstrated for enhanced repair of DNA damage induced by neocarzinostatin in radio-adapted cells. (author)

  9. Genetic profile of Egyptian hepatocellular-carcinoma associated with hepatitis C virus Genotype 4 by 15 K cDNA microarray: Preliminary study

    Mansour Tarek

    2008-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory. Findings We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. Protein phosphatase 3 (PPP3R1 is involved in 10 different pathways followed by fibroblast growth factor receptor 1 (FGFR1, Cas-Br-M ecotropic retroviral transforming sequence b (CBLB, spleen tyrosine kinase (SYK involved in three pathways; bone morphogenetic protein 8a (BMP8A, laminin alpha 3 (LAMA3, cell division cycle 23 (CDC23 involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1 (LTA involved in 3 pathways and alpha-2-macroglobulin (A2M, phosphorylase kinase alpha 2-liver (PHKA2 and MAGI1 membrane associated guanylate kinase 1 (MAGI1 involved in 2 pathways. In addition, 22 genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA. Conclusion This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated

  10. Field-based evidence of fast and global increase of Plasmodium falciparum drug-resistance by DNA-microarrays and PCR/RFLP in Niger

    Ariey Fredéric

    2009-02-01

    Full Text Available Abstract Background Over the last years, significant progress has been made in the comprehension of the molecular mechanism of malaria resistance to drugs. Together with in vivo tests, the molecular monitoring is now part of the survey strategy of the Plasmodium sensitivity. Currently, DNA-microarray analysis allows the simultaneous study of many single nucleotide polymorphisms (SNP of Plasmodium isolates. In December 2005, the International Federation of the Red Cross distributed two million three hundred thousand long-lasting insecticide nets to pregnant women and mothers of under five years children in the whole Niger. Then, Niger adopted artemisinin-based combination therapy as first-line treatment. Methods Thirty four SNPs of pfcrt, pfdhfr, pfdhps, pfmdr and pfATPase were analysed by DNA-microarray and PCR/RFLP in two villages – Zindarou and Banizoumbou – with different durations of malaria transmission. The main objective of the study was to measure the dynamics of Plasmodium falciparum resistant strains and associated factors. Results This study shows a global and clear increase of the drug-resistance associated molecular markers frequencies during a relatively short-time period of four years. Markers associated with resistance to chloroquine and sulphonamids were more frequently found in the short transmission zone than in the long transmission one. The pfcrt76T mutation is significantly more present at Banizoumbou than Zindarou (38.3% vs 25.2%, p = 0.013. This work allowed the screening of several field strains for five SNPs of PfATPase6 gene. The pfATPase6S769N, candidate mutation of resistance to artemisinin was not found. However the pfATPsaeA623E mutation was found in 4.7% of samples. Conclusion A significant increase of several SNPs frequencies was highlighted over a four-year period. The polymorphism of five PfATPase6 gene SNPs was described. The global, large and fast increase of the molecular resistance is discussed in the

  11. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  12. Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB1, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB1-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle

  13. Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

    Geue, Lutz; Stieber, Bettina; Monecke, Stefan; Engelmann, Ines; Gunzer, Florian; Slickers, Peter; Braun, Sascha D.; Ehricht, Ralf

    2014-01-01

    In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and mul...

  14. DNA Microarray and Gene Ontology Enrichment Analysis Reveals That a Mutation in opsX Affects Virulence and Chemotaxis in Xanthomonas oryzae pv. oryzae

    Kim, Hong-Il; Park, Young-Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice (Oryza sativa L.). In this study, we investigated the effect of a mutation in opsX (XOO1056), which encodes a saccharide biosynthesis regulatory protein, on the virulence and bacterial chemotaxis of Xoo. We performed DNA microarray analysis, which showed that 63 of 2,678 genes, including genes related to bacterial motility (flagellar and chemotaxis proteins) were significantly downregulated (<−2 log2 fold changes) by the mutation in opsX. Indeed, motility assays showed that the mutant strain was nonmotile on semisolid agar swarm plates. In addition, a mutant strain (opsX::Tn5) showed decreased virulence against the susceptible rice cultivar, IR24. Quantitative real-time RT-PCR reaction was performed to confirm the expression levels of these genes, including those related to flagella and chemotaxis, in the opsX mutant. Our findings revealed that mutation of opsX affects both virulence and bacterial motility. These results will help to improve our understanding of Xoo and provide insight into Xoo-rice interactions. PMID:27298594

  15. Minimizing DNA microarrays to a single molecule per spot: using zero-mode waveguide technology to obtain kinetic data for a large number of short oligonucleotide hybridization reactions

    Sobek, Jens; Rehrauer, Hubert; Kuhn, Gerrit; Schlapbach, Ralph

    2016-03-01

    We have shown recently that the hybridization of short oligonucleotides can be studied in a zero-mode waveguide nanostructure (ZMW) chip using a modified DNA sequencer.[1] Here we present an extension of this method enabling the parallel measurement of kinetic constants of a large number of hybridization reactions on a single chip. This can be achieved by immobilization of a mixture of oligonucleotides, which leads to a statistical and random distribution of single molecules in the 150'000 ZMWs of a SMRT™ cell. This setup is comparable to a classical microarray with ZMWs in place of spots but unknown allocation of probes. The probe surface density is reduced by a factor of ~1010 allowing the study of hybridization in the absence of interactions with neighboring probes. Hybridization with a dye labelled oligonucleotide results in trains of fluorescence pulses from which interpulse durations (IPDs) and pulse widths (PWs) can be extracted. Since the identity of a probe in a ZMW is unknown, the immobilized oligonucleotide is sequenced in a subsequent step. After mapping the fluorescence traces to the sequence, the association and dissociation rate constant for each oligonucleotide can be calculated. By selecting suitable probes, the method can be used to determine rate constants of hybridization for a large number of mismatch oligonucleotides in a single measurement and at single-molecule level.

  16. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  17. Genes associated with heavy metal tolerance and accumulation in Zn/Cd hyperaccumulator Arabidopsis halleri: a genomic survey with cDNA microarray.

    Chiang, Huai-Chih; Lo, Jing-Chi; Yeh, Kuo-Chen

    2006-11-01

    To survive in variable soil conditions, plants possess homeostatic mechanisms to maintain a suitable concentration of essential heavy metal ions. Certain plants, inhabiting heavy metal-enriched or -contaminated soil, thus are named hyperaccumulators. Studying hyperaccumulators has great potential to provide information for phytoremediation. To better understand the hyperaccumulating mechanism, we used an Arabidopsis cDNA microarray to compare the gene expression of the Zn/Cd hyperaccumulator Arabidopsis halleri and a nonhyperaccumulator, Arabidopsis thaliana. By analyzing the expression of metal-chelators, antioxidation-related genes, and transporters, we revealed a few novel molecular features. We found that metallothionein 2b and 3, APX and MDAR4 in the ascorbate-glutathione pathway, and certain metal transporters in P(1B)-type ATPase, ZIP, Nramp, and CDF families, are expressed at higher levels in A. halleri than in A. thaliana. We further validated that the enzymatic activity of ascorbate peroxidase and class III peroxidases are highly elevated in A. halleri. This observation positively correlates with the higher ability of A. halleri to detoxify H2O2 produced by cadmium and paraquat treatments. We thus suggest that higher peroxidase activities contribute to the heavy metal tolerance in A. halleri by alleviating the ROS damage. We have revealed genes that could be candidates for the future engineering of plants with large biomass for use in phytoremediation. PMID:17144312

  18. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    Railo, Antti [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Pajunen, Antti [Department of Biochemistry, University of Oulu (Finland); Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Vainio, Seppo, E-mail: Seppo.Vainio@oulu.fi [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)

    2009-10-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  19. Profiling of hepatic gene expression of mice fed with edible japanese mushrooms by DNA microarray analysis: comparison among Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus.

    Sato, Mayumi; Tokuji, Yoshihiko; Yoneyama, Shozo; Fujii-Akiyama, Kyoko; Kinoshita, Mikio; Ohnishi, Masao

    2011-10-12

    To compare and estimate the effects of dietary intake of three kinds of mushrooms (Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus), mice were fed a diet containing 10-14% of each mushroom for 4 weeks. Triacylglycerol in the liver and plasma decreased and plasma cholesterol increased in the P. ostreatus-fed group compared with those in the control group. Cholesterol in the liver was lower in the G. frondosa-fed group than in the control group, but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the liver revealed differences of gene expression patterns among mushrooms. Ctp1a and Fabp families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes, such as Mx1, Rsad2, and Oas1, were upregulated. PMID:21910414

  20. DIFFERENTIAL EXPRESSION OF GENES IN OMENTAL FAT OF NORMAL WEIGHT AND OBESE SUBJECTS AND OBESE DIABETIC PATIENTS USING cDNA MICROARRAY

    LUO Tian-hong; ZHENG Pei-zheng; ZHAO Chun-jun; ZHAO Yu; LI Guo; ZHANG Hong-li; LI Wen-yi; LIU You-ping; LUO Min; WANG Kan-kan; ZHANG Ji

    2007-01-01

    Objective To identify genes differentially expressed in omental fat of normal weight subjects,obese subjects and obese diabetic patients. Methods Using a high-density cDNA microarray, gene expression profile of omental fat from normal weigh subjects, obese subjects and obese diabetic patients were compared.Results Totally, 119 and 257 genes were up-regulated in obese subjects and obese diabetic patients respectively,while 46 and 58 genes were down-regulated. A total of 77 genes, including PDK4, which switched from carbohydrate to fatty acids as the primary source of fuel, were up-regulated in both obese and obese diabetic patients, while 8 genes, including key enzymes in lipid synthesis, such as HMG-CoA synthase, fatty acid synthase and stearoyl-CoA desaturase, were down-regulated in both groups. Tyrosine-3-monooxygenase/tryptophan 5-monooxygenase activation protein θ ( YWHAZ) , a negative regulator for insulin signal transduction, was up-regulated only in obese diabetic patient, but not in normal-glycemic obese subjects. Conclusion The study demonstrated that decrease of lipogenesis along with increase of fatty acids oxidation of adipose tissue could be a common cause of insulin resistance in obesity and type 2 diabetes, while block of insulin signal transduction may trigger the transition from obesity to diabetes. Further exploration of these genes will be useful in the understanding of the pathogenesis of obesity and diabetes.

  1. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa

    2006-01-01

    Using a cDNA microarray consisting of 9198 expressed sequence tags, we surveyed the gene expression profiles in shoots and roots of a rice hybrid, Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (P<0.01) expression differences in shoots or roots among the three genotypes, of which 121 showed expression polymorphisms in both shoots and roots, and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohydrate, energy and lipid metabolisms and stress response than expected, while less frequently in categories of amino acid metabolism, transcription and translation regulation, and signal transduction. A total of 214 sequences exhibited significant (P<0.05) mid-parent heterosis in expression, of which 117 had homology to genes with known functions, assigned in the categories of basic metabolism, genetic information processing, cell growth and death, signal transduction, transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation, and for characterizing the molecular mechanism of seedling development and heterosis in rice.

  2. Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP in Brassica napus L.

    Wayne E Clarke

    Full Text Available Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38. The main goal of this project was to combine sequence capture with next generation sequencing (NGS to discover single nucleotide polymorphisms (SNPs in specific areas of the B. napus genome historically associated (via quantitative trait loci -QTL- analysis to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively. Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.

  3. Lower mitochondrial DNA content relates to high-altitude adaptation in Tibetans.

    Li, Yue; Huang, Wei; Yu, Qin; Cheng, Yao-Ting; Kong, Qing-Peng

    2016-01-01

    Mitochondrial DNA (mtDNA) is crucial to mitochondria in energy production and other physiological functions. When lowlanders arrive at high altitude, the mitochondrial content tends to decrease. However, the mtDNA content of native highlanders share the same feature as lowlanders remains unknown. It is also interesting to dissect the other changes in blood plasma that might accompany the change of mtDNA content. To address these issues, we recruited 241 Tibetan subjects in Tibet and 220 Han subjects in Shaanxi province. Relative mtDNA copy number and blood biochemical indexes were measured. Results show that relative mtDNA copy number in Tibetans is significantly lower as compared to Han subjects; sex, age, blood glucose, triglyceride and total cholesterol show no influence on mtDNA content, but carbon dioxide combining power is negatively correlated with mtDNA content. These results indicate that an increase in CO2 combining power along with lower mtDNA content may provide adaptive potential. PMID:24845439

  4. Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels

    Feyereisen R

    2006-06-01

    Full Text Available Abstract Background Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. Results Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76% showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses. Conclusion This analysis of the

  5. Effect and adaptive response of lymphocytes DNA induced by low dose irradiation

    Fluorometric analysis of DNA unwinding (FADU) was conducted and was proved to be an optimal method for studying DNA strand breaks induced by low dose irradiation. The linear dose response curve was obtained. The minimum detected dose was 0.3 Gy. There was no effect of low dose γ-rays (0.5∼8.0 cGy) on DNA strand breaks of quiescent and mitogen-induced lymphocytes. The 0.5∼4.0 cGy γ-rats could induce adaptive response of lymphocytes' DNA strand breaks, especially, at the doses of 2.0 and 4.0 cGy. The challenge doses of 5∼20 Gy could make the adaptive response appearance, and the 15 Gy was the best one. The 3-AB could powerfully inhibit the adaptive response. The repair of DNA strand breaks (37 degree C, 15∼60 min) caused by 15 Gy γ-rays could be promoted by the low dose γ-ray irradiation (2.0 cGy), but no difference was found at 37 degree C, 120 min

  6. Exploring the genes associated with the response to intravenous immunoglobulin in patients with Kawasaki disease using DNA microarray analysis.

    Xing, Yanlin; Wang, Hong; Liu, Xiaomei; Yu, Xianyi; Chen, Rui; Wang, Ce; Yu, Xuexin; Sun, Le

    2015-02-01

    In this study we aimed to screen genes associated with intravenous immunoglobulin (IVIG) responding in patients with Kawasaki disease (KD) and thus explore the underlying molecular mechanism of IVIG resistance. The differentially expressed genes (DEGs) were identified by samr package in R. Then, protein-protein interaction (PPI) networks were constructed by STRING software. We further collected the regulatory data from TRANSFAC database, followed by regulatory interaction network construction. A total 194 of DEGs, including 185 up- and 9 down-regulated DEGs, were identified between IVIG-responding and non-responding patients with KD at acute stage. In contrast, no DEGs were found at convalescent stage. PPI networks and regulatory networks were constructed based on the 185 up-regulated genes at acute stage. The degrees of TFRC (transferrin receptor protein 1) and GADD45A (growth arrest and DNA-damage-inducible alpha) were higher than other genes, and meanwhile MYC (V-Myc Myelocytomatosis Viral Oncogene Homolog) and E2F1 (E2F Transcription Factor 1) were found to be two TFs (transcription factors) with the highest degrees. In conclusions, the response to IVIG in Kawasaki disease patients may be involved in the expression of TFRC, GADD45A, MYC and E2F1. PMID:25449331

  7. Thermal adaptation and clinal mitochondrial DNA variation of European anchovy.

    Silva, Gonçalo; Lima, Fernando P; Martel, Paulo; Castilho, Rita

    2014-10-01

    Natural populations of widely distributed organisms often exhibit genetic clinal variation over their geographical ranges. The European anchovy, Engraulis encrasicolus, illustrates this by displaying a two-clade mitochondrial structure clinally arranged along the eastern Atlantic. One clade has low frequencies at higher latitudes, whereas the other has an anti-tropical distribution, with frequencies decreasing towards the tropics. The distribution pattern of these clades has been explained as a consequence of secondary contact after an ancient geographical isolation. However, it is not unlikely that selection acts on mitochondria whose genes are involved in relevant oxidative phosphorylation processes. In this study, we performed selection tests on a fragment of 1044 bp of the mitochondrial cytochrome b gene using 455 individuals from 18 locations. We also tested correlations of six environmental features: temperature, salinity, apparent oxygen utilization and nutrient concentrations of phosphate, nitrate and silicate, on a compilation of mitochondrial clade frequencies from 66 sampling sites comprising 2776 specimens from previously published studies. Positive selection in a single codon was detected predominantly (99%) in the anti-tropical clade and temperature was the most relevant environmental predictor, contributing with 59% of the variance in the geographical distribution of clade frequencies. These findings strongly suggest that temperature is shaping the contemporary distribution of mitochondrial DNA clade frequencies in the European anchovy. PMID:25143035

  8. Integrated Amplification Microarrays for Infectious Disease Diagnostics

    Darrell P. Chandler

    2012-11-01

    Full Text Available This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

  9. Various Versions of K-means Clustering Algorithm for Segmentation of Microarray Image

    D.Rama Krishna; J Harikiran; Dr.P.V.Lakshmi; Dr.K.V.Ramesh

    2013-01-01

    A Deoxyribonucleic Acid (DNA) microarray is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array. The analysis of DNA microarray images allows the identification of gene expressions to draw biological conclusions for applications ranging from genetic profiling to diagnosis of cancer. The DNA microarray image analysis includes three tasks: gridding, segmentation and intensity extraction. The segmentation step of microarray i...

  10. Phenotypic MicroRNA Microarrays

    Veronica Soloveva; Michel Liuzzi; Jin Yeop Kim; Hi Chul Kim; Jin Yeong Heo; Yong-Jun Kwon

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the bio...

  11. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  12. Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

    Sato, Fumiaki; Tsuchiya, Soken; Terasawa, Kazuya; Tsujimoto, Gozoh

    2009-01-01

    Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platfo...

  13. Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

    2007-01-01

    Pseudoalteromonas sp. SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL-PCR (GenBank accession Nos DQ640312, DQ504163). The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

  14. DNA microarray data integration by ortholog gene analysis reveals potential molecular mechanisms of estrogen-dependent growth of human uterine fibroids

    Shou Jianyong

    2007-04-01

    Full Text Available Abstract Background Uterine fibroids or leiomyoma are a common benign smooth muscle tumor. The tumor growth is well known to be estrogen-dependent. However, the molecular mechanisms of its estrogen-dependency is not well understood. Methods Differentially expressed genes in human uterine fibroids were either retrieved from published papers or from our own statistical analysis of downloaded array data. Probes for the same genes on different Affymetrix chips were mapped based on probe comparison information provided by Affymetrix. Genes identified by two or three array studies were submitted for ortholog analysis. Human and rat ortholog genes were identified by using ortholog gene databases, HomoloGene and TOGA and were confirmed by synteny analysis with MultiContigView tool in the Ensembl genome browser. Results By integrated analysis of three recently published DNA microarray studies with human tissue, thirty-eight genes were found to be differentially expressed in the same direction in fibroid compared to adjacent uterine myometrium by at least two research groups. Among these genes, twelve with rat orthologs were identified as estrogen-regulated from our array study investigating uterine expression in ovariectomized rats treated with estrogen. Functional and pathway analyses of the twelve genes suggested multiple molecular mechanisms for estrogen-dependent cell survival and tumor growth. Firstly, estrogen increased expression of the anti-apoptotic PCP4 gene and suppressed the expression of growth inhibitory receptors PTGER3 and TGFBR2. Secondly, estrogen may antagonize PPARγ signaling, thought to inhibit fibroid growth and survival, at two points in the PPAR pathway: 1 through increased ANXA1 gene expression which can inhibit phospholipase A2 activity and in turn decrease arachidonic acid synthesis, and 2 by decreasing L-PGDS expression which would reduce synthesis of PGJ2, an endogenous ligand for PPARγ. Lastly, estrogen affects retinoic

  15. Role of Cell Cycle Regulation and MLH1, A Key DNA Mismatch Repair Protein, In Adaptive Survival Responses. Final Report

    David A. Boothman

    1999-08-11

    Due to several interesting findings on both adaptive survival responses (ASRs) and DNA mismatch repair (MMR), this grant was separated into two discrete Specific Aim sets (each with their own discrete hypotheses). The described experiments were simultaneously performed.

  16. Multicenter Evaluation of a New DNA Microarray for Rapid Detection of Clinically Relevant bla Genes from ? -Lactam-Resistant Gram-Negative Bacteria

    Bogaerts, P.; Hujer, A.M.; Naas, T.; de Castro, R. R.; A. Endimiani; Nordmann, P.; Glupczynski, Y.; Bonomo, R.A.

    2011-01-01

    A new commercial low-density microarray which identifies common extended-spectrum β-lactamase plasmid-mediated cephalosporinase genes, as well as carbapenemase (blaKPC and blaNDM) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceae possessing different bla genes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated blaAmpC, blaKPC, and blaNDM genes using bla gene sequencing as the re...

  17. Universal ligation-detection-reaction microarray applied for compost microbes

    Romantschuk Martin

    2008-12-01

    Full Text Available Abstract Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.

  18. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S; Golova, Julia; Perov, Alexander; Protic, Miroslava; Robison, Richard; Schipma, Matthew; White, Amanda; Willse, Alan

    2006-01-01

    A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

  19. DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

    Piontkivska Helen

    2005-02-01

    Full Text Available Abstract Background Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes. Results DNA repeat arrays in the sequenced portion of the chicken (Gallus gallus autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human. Conclusions The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.

  20. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA – Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    Hatt, Lotte; Aagaard, Mads M.; Graakjaer, Jesper; Bach, Cathrine; Sommer, Steffen; Inge E Agerholm; Kølvraa, Steen; Bojesen, Anders

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylati...

  1. Simulation of DNA damage clustering after proton irradiation using an adapted DBSCAN algorithm.

    Francis, Ziad; Villagrasa, Carmen; Clairand, Isabelle

    2011-03-01

    In this work the "Density Based Spatial Clustering of Applications with Noise" (DBSCAN) algorithm was adapted to early stage DNA damage clustering calculations. The resulting algorithm takes into account the distribution of energy deposit induced by ionising particles and a damage probability function that depends on the total energy deposit amount. Proton track simulations were carried out in small micrometric volumes representing small DNA containments. The algorithm was used to determine the damage concentration clusters and thus to deduce the DSB/SSB ratios created by protons between 500keV and 50MeV. The obtained results are compared to other calculations and to available experimental data of fibroblast and plasmid cells irradiations, both extracted from literature. PMID:21232812

  2. DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

    Kim, Mee Kum; Oh, Joo Youn; Ko, Jung Hwa; Lee, Hyun Ju; Jung, Jin Ho; Wee, Won Ryang; Lee, Jin Hak; Park, Chung-Gyu; Kim, Sang Joon; Ahn, Curie; Kim, Seung-Jun; Hwang, Seung Yong

    2009-01-01

    Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray metho...

  3. DNA microarray for simultaneous screening and detection of seven rickettsia%7种立克次体甄别检测基因芯片方法的建立

    李灵云; 张英杰; 王升启; 刘琪琦

    2015-01-01

    目的:建立一种能同时检测7种立克次体的化学发光基因芯片法。方法根据NCBI公开发表的7种立克次体的序列设计引物和探针,制备立克次体甄别检测基因芯片。利用多重不对称PCR法扩增立克次体靶基因片段,标记的产物与基因芯片上的探针杂交,经清洗、化学发光显色后进行结果分析。在优化的多重PCR体系、杂交反应和化学发光检测条件下,评价芯片的特异性、灵敏度、重复性。用实时荧光PCR法与芯片法分别检测莫氏立克次体梯度稀释的核酸,比较两种方法的灵敏度。制备双盲模拟样本,进一步评价芯片方法的准确性。结果该研究共筛选出1对通用引物、4对特异性引物和1条立克次体属通用探针、9条特异性检测探针。该芯片检测质粒DNA的灵敏度为1.5×102~3×103拷贝/反应,检测模拟样本的灵敏度为103~104拷贝/μl。实时荧光PCR法与芯片法检测结果一致,实时荧光PCR法比芯片法灵敏度高10倍。双盲模拟样本检测符合率为100%。结论成功建立了可同时检测7种立克次体的化学发光基因芯片检测方法,为立克次体病的临床诊断和流行病学调查提供了一种新的高通量检测手段。%Objective To develop a chemiluminescence ( CL ) imaging DNA microarray method for simultaneous detection of seven rickettsiae.Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes.The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae.The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method, and then hybridized with microarray that was scanned after washing and chemiluminescence coloration, before the results were analyzed.Facilitated by the optimization of the multiplex PCR system, hybridization, and chemiluminescence imagination, we evaluated the

  4. Phenotypic MicroRNA Microarrays

    Veronica Soloveva

    2013-04-01

    Full Text Available Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  5. The Impact of Photobleaching on Microarray Analysis

    Marcel von der Haar

    2015-09-01

    Full Text Available DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results.

  6. Ethanol extract of Hedyotis diffusa willd upregulates G0/G1 phase arrest and induces apoptosis in human leukemia cells by modulating caspase cascade signaling and altering associated genes expression was assayed by cDNA microarray.

    Kuo, Yu-Jui; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-09-01

    The authors' previous study has shown that water extract of Hedyotis diffusa Willd (HDW) promoted immune response and exhibited anti-leukemic activity in BALB/c leukemic mice in vivo. In this study, the anti-proliferation effects of ethanol extract of H. diffusa Willd (EEHDW) on lung cancer cell lines (A549, H1355, and LLC), leukemia cell lines (HL-60, WEHI-3), and a mouse melanoma cell line (B16F10) in vitro were investigated. The results demonstrated that EEHDW suppressed the cell proliferation of A549, H1355, HL-60, WEHI-3, and B16F10 cells as well as reduced cell viability in a concentration-dependent manner. We found that EEHDW inhibited the cell proliferation of HL-60 cells in concentration-dependent manner. In addition, EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and sub-G1 population (apoptotic cells). EEHDW provoked DNA condensation and DNA damage in HL-60 cells. The activities of caspase-3, caspase-8, and caspase-9 were elevated in EEHDW-treated HL-60 cells. DNA microarray to investigate and display the gene levels related to cell growth, signal transduction, apoptosis, cell adhesion, cell cycle, DNA damage and repair, transcription and translation was also used. These findings suggest that EEHDW may be a potential herbal medicine and therapeutic agent for the treatment of leukemia. PMID:24677778

  7. ProCAT: a data analysis approach for protein microarrays

    Zhu, Xiaowei; Gerstein, Mark; Snyder, Michael

    2006-01-01

    Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approac...

  8. Genome Array on Differentially Expressed Genes of Skin Tissue in Cashmere Goat at Early Anagen of Cashmere Growth Cycle Using DNA Microarray

    DI Jiang; Marzeya Yasen; XU Xin-ming; Lazate Ainiwaer; ZHANG Yan-hua; TIAN Ke-chuan; YU Li-juan; WU Wei-wei; Hanikezi Tulafu; FU Xue-feng

    2014-01-01

    In order to study the molecular mechanism involved in cashmere regeneration, this study investigated the gene expression proifle of skin tissue at various stages of the cashmere growth cycle and screen differentially expressed genes at proangen in 10 cashmere goats at 2 years of age using agilent sheep oligo microarray. Signiifcance analysis of microarray (SAM) methods was used to identify the differentially expressed genes, Hierarchical clustering was performed to clarify these genes in association with different cashmere growth stages, and GO (Gene ontology) and the pathway analyses were con-ducted by a free web-based Molecular Annotation System3.0 (MAS 3.0). Approximately 10 200 probe sets were detected in skin tissue of 2-yr-old cashmere goat. After SAM analysis of the microarray data, totally 417 genes were shown to be differentially expressed at different cashmere growth stages, and 24 genes are signiifcantly up-regulated (21) or down-regulated (3) at proangen concurrently compared to angen and telogen. Hierarchical clustering analysis clearly distinguished the differentially expressed genes of each stage. GO analysis indicated that these altered genes at proangen were predominantly involved in collagen ifbril organization, integrin-mediated signaling pathway, cell-matrix adhesion, cell adhesion, transforming growth factor-β (TGF-β) receptor signaling pathway, regulation of cell growth. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that the signiifcant pathways involved mainly included focal adhesion and extracellular matrixc (ECM)-receptor interaction. Some important genes involved in these biological processes, such as COL1A1, COL1A2, COL3A1, SPARC, CYR61 and CTGF, were related to tissue remolding and repairing and detected by more than one probe with similar expression trends at different stages of cashmere growth cycle. The different expression of these genes may contribute to understanding the molecular mechanism of cashmere

  9. Mathematical design of prokaryotic clone-based microarrays

    Pieterse, B.; Quirijns, E.J.; Schuren, F.H.J.; Werf, M.J. van der

    2005-01-01

    Background: Clone-based microarrays, on which each spot represents a random genomic fragment, are a good alternative to open reading frame-based microarrays, especially for microorganisms for which the complete genome sequence is not available. Since the generation of a genomic DNA library is a rand

  10. Mouse retinal adaptive response to proton irradiation: Correlation with DNA repair and photoreceptor cell death

    Tronov, V. A.; Vinogradova, Yu. V.; Poplinskaya, V. A.; Nekrasova, E. I.; Ostrovsky, M. A.

    2015-01-01

    Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous study revealed non-linear dose-response relationship for cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In present study we used the mouse model for MNU-induced retinal degeneration to evaluate adaptive response of retina to proton irradiation and implication in it of glial Muller cells. The data showed that the recovery of retina after genotoxic agents has been associated with increased efficacy of DNA damage repair and lowered death of retinal photoreceptor cells.

  11. Biotechnology and DNA vaccines for aquatic animals

    Kurath, G.

    2008-01-01

    Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.

  12. Adaptive response in mice exposed to 900 MHz radiofrequency fields: primary DNA damage.

    Bingcheng Jiang

    Full Text Available The phenomenon of adaptive response (AR in animal and human cells exposed to ionizing radiation is well documented in scientific literature. We have examined whether such AR could be induced in mice exposed to non-ionizing radiofrequency fields (RF used for wireless communications. Mice were pre-exposed to 900 MHz RF at 120 µW/cm(2 power density for 4 hours/day for 1, 3, 5, 7 and 14 days and then subjected to an acute dose of 3 Gy γ-radiation. The primary DNA damage in the form of alkali labile base damage and single strand breaks in the DNA of peripheral blood leukocytes was determined using the alkaline comet assay. The results indicated that the extent of damage in mice which were pre-exposed to RF for 1 day and then subjected to γ-radiation was similar and not significantly different from those exposed to γ-radiation alone. However, mice which were pre-exposed to RF for 3, 5, 7 and 14 days showed progressively decreased damage and was significantly different from those exposed to γ-radiation alone. Thus, the data indicated that RF pre-exposure is capable of inducing AR and suggested that the pre-exposure for more than 4 hours for 1 day is necessary to elicit such AR.

  13. DNA microarray for detection of unknown virus in clinical specimens%临床标本中未知病毒基因芯片检测方法探索

    郑夔; 王洪敏; 黄吉城; 夏文英; 洪烨; 李小波; 师永霞; 幸芦琴; 郭波旋

    2011-01-01

    Objective To develop a DNA microarray for detection of unknown virus in clinical specimens. Methods Probes for dengue virus type 1-4 were designed and synthesized, and DNA microarray of dengue virus was prepared. After viral RNA of dengue virus type 1-4 standard strains were extracted, whole genome amplification was performed by using phi29 DNA polymerase and a random primer contain tag sequence, then the genome was amplified and labeled by random PCR with a Cy3 - conjugated tag sequence primer. The fluorescein labeled amplicon were subjected to hybridization with the DNA microarray of dengue virus. Whereafter the novel method was verified by using serum samples collected from pateints with dengue fever in Guangzhou Baiyun airport entry and exit inspection and quarantine frontier port. Results Hybridization signals of dengue virus type 1-4 standard strains were significantly distinguished, and all of the related strains showed a high probe positive rate of 100%. Similarly, 3 serum samples of dengue virus from clinical patients also had significant hybridization signals with very low background noise. Dengue virus types 1, 2 and 3 carried in the 3 serum samples can be clearly identified by the high probe positive rate of 100% related to the microarray. Conclusion The innovative DNA microarray assay can be used to detect dengue virus in clinical specimens. If more specific probes for different species of pathogens are designed and appended to this new DNA microarray, it also can be expanded for unknown pathogens identification in clinical specimens.%目的 探索可用于检测临床标本中未知病毒的基因芯片技术.方法 设计合成1~4型登革病毒基因芯片探针,制备成登革病毒基因芯片.提取1~4型登革病毒标准毒株的核酸RNA,以phi29 DNA聚合酶结合带标签序列的随机引物进行全基因组扩增,再以Cy3荧光染料标记的标签序列引物进行PCR随机扩增标记,标记产物进一步用登革病毒芯片进

  14. An overview of innovations and industrial solutions in Protein Microarray Technology.

    Gupta, Shabarni; Manubhai, K P; Kulkarni, Vishwesh; Srivastava, Sanjeeva

    2016-04-01

    The complexity involving protein array technology reflects in the fact that instrumentation and data analysis are subject to change depending on the biological question, technical compatibility of instruments and software used in each experiment. Industry has played a pivotal role in establishing standards for future deliberations in sustenance of these technologies in the form of protein array chips, arrayers, scanning devices, and data analysis software. This has enhanced the outreach of protein microarray technology to researchers across the globe. These have encouraged a surge in the adaptation of "nonclassical" approaches such as DNA-based protein arrays, micro-contact printing, label-free protein detection, and algorithms for data analysis. This review provides a unique overview of these industrial solutions available for protein microarray based studies. It aims at assessing the developments in various commercial platforms, thus providing a holistic overview of various modalities, options, and compatibility; summarizing the journey of this powerful high-throughput technology. PMID:27089056

  15. Genomic-Wide Analysis with Microarrays in Human Oncology

    Kenichi Inaoka

    2015-10-01

    Full Text Available DNA microarray technologies have advanced rapidly and had a profound impact on examining gene expression on a genomic scale in research. This review discusses the history and development of microarray and DNA chip devices, and specific microarrays are described along with their methods and applications. In particular, microarrays have detected many novel cancer-related genes by comparing cancer tissues and non-cancerous tissues in oncological research. Recently, new methods have been in development, such as the double-combination array and triple-combination array, which allow more effective analysis of gene expression and epigenetic changes. Analysis of gene expression alterations in precancerous regions compared with normal regions and array analysis in drug-resistance cancer tissues are also successfully performed. Compared with next-generation sequencing, a similar method of genome analysis, several important differences distinguish these techniques and their applications. Development of novel microarray technologies is expected to contribute to further cancer research.

  16. Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli.

    Sugimoto, Shinya; Nakayama, Jiro; Fukuda, Daisuke; Sonezaki, Shino; Watanabe, Maki; Tosukhowong, Amonlaya; Sonomoto, Kenji

    2003-01-01

    Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli. PMID:16233497

  17. AffyMiner: mining differentially expressed genes and biological knowledge in GeneChip microarray data

    Xia Yuannan; Nguyen The V; Lu Guoqing; Fromm Michael

    2006-01-01

    Abstract Background DNA microarrays are a powerful tool for monitoring the expression of tens of thousands of genes simultaneously. With the advance of microarray technology, the challenge issue becomes how to analyze a large amount of microarray data and make biological sense of them. Affymetrix GeneChips are widely used microarrays, where a variety of statistical algorithms have been explored and used for detecting significant genes in the experiment. These methods rely solely on the quanti...

  18. Unraveling the rat blood genome-wide transcriptome after oral administration of lavender oil by a two-color dye-swap DNA microarray approach

    Motohide Hori

    2016-06-01

    Full Text Available Lavender oil (LO is a commonly used essential oil in aromatherapy as non-traditional medicine. With an aim to demonstrate LO effects on the body, we have recently established an animal model investigating the influence of orally administered LO in rat tissues, genome-wide. In this brief, we investigate the effect of LO ingestion in the blood of rat. Rats were administered LO at usual therapeutic dose (5 mg/kg in humans, and following collection of the venous blood from the heart and extraction of total RNA, the differentially expressed genes were screened using a 4 × 44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA in conjunction with a two-color dye-swap approach. A total of 834 differentially expressed genes in the blood were identified: 362 up-regulated and 472 down-regulated. These genes were functionally categorized using bioinformatics tools. The gene expression inventory of rat blood transcriptome under LO, a first report, has been deposited into the Gene Expression Omnibus (GEO: GSE67499. The data will be a valuable resource in examining the effects of natural products, and which could also serve as a human model for further functional analysis and investigation.

  19. Unraveling the rat blood genome-wide transcriptome after oral administration of lavender oil by a two-color dye-swap DNA microarray approach.

    Hori, Motohide; Kubo, Hiroko; Shibato, Junko; Saito, Tomomi; Ogawa, Tetsuo; Wakamori, Minoru; Masuo, Yoshinori; Shioda, Seiji; Rakwal, Randeep

    2016-06-01

    Lavender oil (LO) is a commonly used essential oil in aromatherapy as non-traditional medicine. With an aim to demonstrate LO effects on the body, we have recently established an animal model investigating the influence of orally administered LO in rat tissues, genome-wide. In this brief, we investigate the effect of LO ingestion in the blood of rat. Rats were administered LO at usual therapeutic dose (5 mg/kg) in humans, and following collection of the venous blood from the heart and extraction of total RNA, the differentially expressed genes were screened using a 4 × 44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a two-color dye-swap approach. A total of 834 differentially expressed genes in the blood were identified: 362 up-regulated and 472 down-regulated. These genes were functionally categorized using bioinformatics tools. The gene expression inventory of rat blood transcriptome under LO, a first report, has been deposited into the Gene Expression Omnibus (GEO): GSE67499. The data will be a valuable resource in examining the effects of natural products, and which could also serve as a human model for further functional analysis and investigation. PMID:27330992

  20. Investigating the global genomic diversity of Escherichia coli using a multi-genome DNA microarray platform with novel gene prediction strategies

    LeClerc Joseph E

    2011-07-01

    Full Text Available Abstract Background The gene content of a diverse group of 183 unique Escherichia coli and Shigella isolates was determined using the Affymetrix GeneChip® E. coli Genome 2.0 Array, originally designed for transcriptome analysis, as a genotyping tool. The probe set design utilized by this array provided the opportunity to determine the gene content of each strain very accurately and reliably. This array constitutes 10,112 independent genes representing four individual E. coli genomes, therefore providing the ability to survey genes of several different pathogen types. The entire ECOR collection, 80 EHEC-like isolates, and a diverse set of isolates from our FDA strain repository were included in our analysis. Results From this study we were able to define sets of genes that correspond to, and therefore define, the EHEC pathogen type. Furthermore, our sampling of 63 unique strains of O157:H7 showed the ability of this array to discriminate between closely related strains. We found that individual strains of O157:H7 differed, on average, by 197 probe sets. Finally, we describe an analysis method that utilizes the power of the probe sets to determine accurately the presence/absence of each gene represented on this array. Conclusions These elements provide insights into understanding the microbial diversity that exists within extant E. coli populations. Moreover, these data demonstrate that this novel microarray-based analysis is a powerful tool in the field of molecular epidemiology and the newly emerging field of microbial forensics.

  1. DNA甲基化微阵列的非参数贝叶斯聚类算法%Nonparametric Bayesian Clustering Methods of DNA Methylation Microarray

    张林; 刘辉

    2012-01-01

    A model based clustering algorithm for Illumina GoldenGate microarray data is proposed in this paper. By infinite beta mixture model and by adopting Dirichlet process as prior knowledge, the cluster structure can be denned based on model and data. Simulation results demonstrate that this methodology can estimate the number of clusters, the cluster mixing weight and the own characteristic of each cluster, and can reach relatively ideal clustering effect.%面向Illumina GoldenGate甲基化微阵列数据提出了一种基于模型的聚类算法.算法通过建立贝塔无限混合模型,采用Dirichlet过程作为先验,实现了基于数据和模型的聚类结构的建立,实验结果表明该算法能够有效估计出聚类类别个数、每个聚类类别的混合权重、每个聚类类别的特征等信息,达到比较理想的聚类效果.

  2. Alteration of Gene Expression Profile in Kidney of Spontaneously Hypertensive Rats Treated with Protein Hydrolysate of Blue Mussel (Mytilus edulis) by DNA Microarray Analysis.

    Feng, Junli; Dai, Zhiyuan; Zhang, Yanping; Meng, Lu; Ye, Jian; Ma, Xuting

    2015-01-01

    Marine organisms are rich sources of bioactive components, which are often reported to have antihypertensive effects. However, the underlying mechanisms have yet to be fully identified. The aim of this study was to investigate the antihypertensive effect of enzymatic hydrolysis of blue mussel protein (HBMP) in rats. Peptides with in vitro ACE inhibitory activity were purified from HBMP by ultrafiltration, gel filtration chromatography and reversed-phase high performance liquid chromatography. And the amino acid sequences of isolated peptides were estimated to be Val-Trp, Leu-Gly-Trp, and Met-Val-Trp-Thr. To study its in vivo action, spontaneously hypertensive rats (SHRs) were orally administration with high- or low-dose of HBMP for 28 days. Major components of the renin-angiotensin (RAS) system in serum of SHRs from different groups were analyzed, and gene expression profiling were performed in the kidney of SHRs, using the Whole Rat Genome Oligonucleotide Microarray. Results indicated although genes involved in RAS system were not significantly altered, those related to blood coagulation system, cytokine and growth factor, and fatty acids metabolism were remarkablely changed. Several genes which were seldom reported to be implicated in pathogenesis of hypertension also showed significant expression alterations after oral administration of HBMP. These data provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of HBMP, and will contribute towards increased value-added utilization of blue mussel protein. PMID:26517713

  3. Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

    Chen, Fangyi; Tang, Qi; Bian, Ke; Humulock, Zachary T; Yang, Xuedong; Jost, Marco; Drennan, Catherine L; Essigmann, John M; Li, Deyu

    2016-04-18

    The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. PMID:26919079

  4. Application of subtracted gDNA microarray-assisted Bulked Segregant Analysis for rapid discovery of molecular markers associated with day-neutrality in strawberry (Fragaria x ananassa).

    Gor, Mian Chee; Mantri, Nitin; Pang, Edwin

    2016-01-01

    A Fragaria Discovery Panel (FDP; strawberry-specific SDA) containing 287 features was constructed by subtracting the pooled gDNA of nine non-angiosperm species from the pooled gDNA of five strawberry genotypes. This FDP was used for Bulk Segregant Analysis (BSA) to enable identification of molecular markers associated with day-neutrality. Analysis of hybridisation patterns of a short day (SD) DNA bulk and three day-neutral (DN) DNA bulks varying in flowering strength allowed identification of a novel feature, FaP2E11, closely linked to CYTOKININ OXIDASE 1 (CKX1) gene possibly involved in promoting flowering under non-inductive condition. The signal intensities of FaP2E11 feature obtained from the strong DN bulk (DN1) is three fold higher than the short day bulk (SD), indicating that the putative marker may linked to a CKX1 variant allele with lower enzyme activity. We propose a model for flowering regulation based on the hypothesis that flowering strength may be regulated by the copy number of FaP2E11-linked CKX1 alleles. This study demonstrates the feasibility of the SDA-based BSA approach for the identification of molecular markers associated with day-neutrality in strawberry. This innovative strategy is an efficient and cost-effective approach for molecular marker discovery. PMID:27586242

  5. Towards standardization of microarray-based genotyping of Salmonella

    Löfström, Charlotta; Grønlund, Hugo Ahlm; Riber, Leise; Vigre, Håkan; Folling, Liselotte; Huehn, Stephan; Malorny, Burkhard; Rådström, Peter; Rudi, Knut; Hoorfar, Jeffrey

    2010-01-01

    Genotyping is becoming an increasingly important tool to improve risk assessments of Salmonella. DNA microarray technology is a promising diagnostic tool that can provide high resolution genomic profile of many genes simultaneously. However, standardization of DNA microarray analysis is needed...... before it can be used as a tool in source attribution models for comparable characterization of isolates across laboratories and countries. The reproducibility of data was evaluated for a simple and single-dye DNA microarray (Huehn et al., Appl Environ Microbiol, 2009, 75:1011-1020) for genotyping of...... agreement (Kappa = 0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this as the most critical factor for standardization between laboratories. In conclusion, this study indicates that it is possible to set up an international standard for a...

  6. Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

    Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit; Al Husini, Nadra; Dhoondia, Zuzer; Ansari, Athar; Pascuzzi, Pete E; Tran, Elizabeth J

    2016-02-01

    Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. PMID:26833086

  7. The use of microarrays in microbial ecology

    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

  8. Identification of Novel Protein–Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a...

  9. Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity.

    Mori, Tsukasa; Hiraka, Ikuei; Kurata, Youichi; Kawachi, Hiroko; Mano, Nobuhiro; Devlin, Robert H; Nagoya, Hiroyuki; Araki, Kazuo

    2007-03-01

    Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F(2) and F(3) generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Delta6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F(2) and F(3) salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F(2) and F(3) fish were completely

  10. rapmad: Robust analysis of peptide microarray data

    Rothermel Andrée

    2011-08-01

    Full Text Available Abstract Background Peptide microarrays offer an enormous potential as a screening tool for peptidomics experiments and have recently seen an increased field of application ranging from immunological studies to systems biology. By allowing the parallel analysis of thousands of peptides in a single run they are suitable for high-throughput settings. Since data characteristics of peptide microarrays differ from DNA oligonucleotide microarrays, computational methods need to be tailored to these specifications to allow a robust and automated data analysis. While follow-up experiments can ensure the specificity of results, sensitivity cannot be recovered in later steps. Providing sensitivity is thus a primary goal of data analysis procedures. To this end we created rapmad (Robust Alignment of Peptide MicroArray Data, a novel computational tool implemented in R. Results We evaluated rapmad in antibody reactivity experiments for several thousand peptide spots and compared it to two existing algorithms for the analysis of peptide microarrays. rapmad displays competitive and superior behavior to existing software solutions. Particularly, it shows substantially improved sensitivity for low intensity settings without sacrificing specificity. It thereby contributes to increasing the effectiveness of high throughput screening experiments. Conclusions rapmad allows the robust and sensitive, automated analysis of high-throughput peptide array data. The rapmad R-package as well as the data sets are available from http://www.tron-mz.de/compmed.

  11. Adapt

    Bargatze, L. F.

    2015-12-01

    Active Data Archive Product Tracking (ADAPT) is a collection of software routines that permits one to generate XML metadata files to describe and register data products in support of the NASA Heliophysics Virtual Observatory VxO effort. ADAPT is also a philosophy. The ADAPT concept is to use any and all available metadata associated with scientific data to produce XML metadata descriptions in a consistent, uniform, and organized fashion to provide blanket access to the full complement of data stored on a targeted data server. In this poster, we present an application of ADAPT to describe all of the data products that are stored by using the Common Data File (CDF) format served out by the CDAWEB and SPDF data servers hosted at the NASA Goddard Space Flight Center. These data servers are the primary repositories for NASA Heliophysics data. For this purpose, the ADAPT routines have been used to generate data resource descriptions by using an XML schema named Space Physics Archive, Search, and Extract (SPASE). SPASE is the designated standard for documenting Heliophysics data products, as adopted by the Heliophysics Data and Model Consortium. The set of SPASE XML resource descriptions produced by ADAPT includes high-level descriptions of numerical data products, display data products, or catalogs and also includes low-level "Granule" descriptions. A SPASE Granule is effectively a universal access metadata resource; a Granule associates an individual data file (e.g. a CDF file) with a "parent" high-level data resource description, assigns a resource identifier to the file, and lists the corresponding assess URL(s). The CDAWEB and SPDF file systems were queried to provide the input required by the ADAPT software to create an initial set of SPASE metadata resource descriptions. Then, the CDAWEB and SPDF data repositories were queried subsequently on a nightly basis and the CDF file lists were checked for any changes such as the occurrence of new, modified, or deleted

  12. In vitro antioxidant and antigenotoxic potentials of myricetin-3-o-galactoside and myricetin-3-o-rhamnoside from Myrtus communis: modulation of expression of genes involved in cell defence system using cDNA microarray.

    Hayder, Nawel; Bouhlel, Ines; Skandrani, Ines; Kadri, Malika; Steiman, Régine; Guiraud, Pascale; Mariotte, Anne-Marie; Ghedira, Kamel; Dijoux-Franca, Marie-Geneviève; Chekir-Ghedira, Leila

    2008-04-01

    Antioxidant activity of myricetin-3-o-galactoside and myricetin-3-o-rhamnoside, isolated from the leaves of Myrtus communis, was determined by the ability of each compound to inhibit xanthine oxidase activity, lipid peroxidation and to scavenge the free radical 1,1-diphenyl-2-picrylhydrazyl. Antimutagenic activity was assessed using the SOS chromotest and the Comet assay. The IC50 values of lipid peroxidation by myricetin-3-o-galactoside and myricetin-3-o-rhamnoside are respectively 160 microg/ml and 220 microg/ml. At a concentration of 100 microg/ml, the two compounds showed the most potent inhibitory effect of xanthine oxidase activity by respectively, 57% and 59%. Myricetin-3-o-rhamnoside was a very potent radical scavenger with an IC50 value of 1.4 microg/ml. Moreover, these two compounds induced an inhibitory activity against nifuroxazide, aflatoxine B1 and H2O2 induced mutagenicity. The protective effect exhibited by these molecules was also determined by analysis of gene expression as response to an oxidative stress using a cDNA micro-array. Myricetin-3-o-galactoside and myricetin-3-o-rhamnoside modulated the expression patterns of cellular genes involved in oxidative stress, respectively (GPX1, TXN, AOE372, SEPW1, SHC1) and (TXNRD1, TXN, SOD1 AOE372, SEPW1), in DNA damaging repair, respectively (XPC, LIG4, RPA3, PCNA, DDIT3, POLD1, XRCC5, MPG) and (TDG, PCNA, LIG4, XRCC5, DDIT3, MSH2, ERCC5, RPA3, POLD1), and in apoptosis (PARP). PMID:18222061

  13. The Porcelain Crab Transcriptome and PCAD, the Porcelain Crab Microarray and Sequence Database

    Tagmount, Abderrahmane; Wang, Mei; Lindquist, Erika; Tanaka, Yoshihiro; Teranishi, Kristen S.; Sunagawa, Shinichi; Wong, Mike; Stillman, Jonathon H.

    2010-01-27

    Background: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. Methodology/Principal Findings: A set of ~;;30K unique sequences (UniSeqs) representing ~;;19K clusters were generated from ~;;98K high quality ESTs from a set of tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66percent of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases.Conclusions/Significance: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in

  14. Identification of prior candidate genes for Sclerotinia local resistance in Brassica napus using Arabidopsis cDNA microarray and Brassica-Arabidopsis comparative mapping

    LIU; Renhu; ZHAO; Jianwei; XIAO; Yong; MENG; Jinling

    2005-01-01

    Arabidopsis cDNA arrays were used to screen the local-defense-associated genes in oilseed rape (Brassica napus L.) at the challenge of Sclerotinia sclerotiorum. 61 genes with two-fold expression changes were screened out from the local tissue around the necrosis. Among them, 36 unique genes were up-regulated and 25 unique genes were down-regulated. RT-PCR and Northern blot results were consistent with the array results, suggesting Arabidopsis arrays were useful for transcriptional profiling of B. napus genes. Some of these genes were located in the interval of some QTLs for Sclerotinia resistance in B. napus by Brassica- Arabidopsis comparative mapping. These genes may have priority to be pursued for more intensive research.

  15. Microarrays for Pathogen Detection and Analysis

    McLoughlin, Kevin S.

    2011-01-01

    DNA microarrays have emerged as a viable platform for detection of pathogenic organisms in clinical and environmental samples. These microbial detection arrays occupy a middle ground between low cost, narrowly focused assays such as multiplex PCR and more expensive, broad-spectrum technologies like high-throughput sequencing. While pathogen detection arrays have been used primarily in a research context, several groups are aggressively working to develop arrays for clinical diagnostics, food ...

  16. Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

    Malloch Gaynor

    2007-11-01

    Full Text Available Abstract Background The green peach aphid, Myzus persicae (Sulzer, is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs. Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection. The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible

  17. Improving comparability between microarray probe signals by thermodynamic intensity correction

    Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka;

    2007-01-01

    different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities into...

  18. Emerging Use of Gene Expression Microarrays in Plant Physiology

    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 e...

  19. Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench).

    Yabe, Shiori; Hara, Takashi; Ueno, Mariko; Enoki, Hiroyuki; Kimura, Tatsuro; Nishimura, Satoru; Yasui, Yasuo; Ohsawa, Ryo; Iwata, Hiroyoshi

    2014-12-01

    For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information. PMID:25914583

  20. Protein microarrays for systems biology

    Lina Yang; Shujuan Guo; Yang Li; Shumin Zhou; Shengce Tao

    2011-01-01

    Systems biology holds the key for understanding biological systems on a system level. It eventually holds the key for the treatment and cure of complex diseases such as cancer,diabetes, obesity, mental disorders, and many others. The '-omics' technologies, such as genomics, transcriptomics,proteomics, and metabonomics, are among the major driving forces of systems biology. Featured as highthroughput, miniaturized, and capable of parallel analysis,protein microarrays have already become an important technology platform for systems biology, In this review, we will focus on the system level or global analysis of biological systems using protein microarrays. Four major types of protein microarrays will be discussed: proteome microarrays, antibody microarrays, reverse-phase protein arrays,and lectin microarrays. We will also discuss the challenges and future directions of protein microarray technologies and their applications for systems biology. We strongly believe that protein microarrays will soon become an indispensable and invaluable tool for systems biology.

  1. Microarray technology and its applications

    Müller, UR

    2006-01-01

    It presents detailed overviews of the different techniques of fabricating microarrays, of the chemistries and preparative steps involved, of the different types of microarrays, and of the instrumentation and optical issues involved.

  2. A non-parametric meta-analysis approach for combining independent microarray datasets: application using two microarray datasets pertaining to chronic allograft nephropathy

    Archer Kellie J; Mas Valeria; Kong Xiangrong

    2008-01-01

    Abstract Background With the popularity of DNA microarray technology, multiple groups of researchers have studied the gene expression of similar biological conditions. Different methods have been developed to integrate the results from various microarray studies, though most of them rely on distributional assumptions, such as the t-statistic based, mixed-effects model, or Bayesian model methods. However, often the sample size for each individual microarray experiment is small. Therefore, in t...

  3. Evaluating different methods of microarray data normalization

    Ferreira Carlos

    2006-10-01

    Full Text Available Abstract Background With the development of DNA hybridization microarray technologies, nowadays it is possible to simultaneously assess the expression levels of thousands to tens of thousands of genes. Quantitative comparison of microarrays uncovers distinct patterns of gene expression, which define different cellular phenotypes or cellular responses to drugs. Due to technical biases, normalization of the intensity levels is a pre-requisite to performing further statistical analyses. Therefore, choosing a suitable approach for normalization can be critical, deserving judicious consideration. Results Here, we considered three commonly used normalization approaches, namely: Loess, Splines and Wavelets, and two non-parametric regression methods, which have yet to be used for normalization, namely, the Kernel smoothing and Support Vector Regression. The results obtained were compared using artificial microarray data and benchmark studies. The results indicate that the Support Vector Regression is the most robust to outliers and that Kernel is the worst normalization technique, while no practical differences were observed between Loess, Splines and Wavelets. Conclusion In face of our results, the Support Vector Regression is favored for microarray normalization due to its superiority when compared to the other methods for its robustness in estimating the normalization curve.

  4. 嗜肺军团菌15种血清型基因芯片检测方法的建立%Development of a DNA Microarray for Detection and Identification of 15 Distinct O Antigen Forms of Legionella Pneumophila

    吴冬雪; 王乃福; 黄晨; 高旗利; 关淳

    2014-01-01

    To establish a rapid, accurate detection method for 15 distinct O antigen forms of Legionella pneumophila using DNA microarray combined with multiplex PCR. The special genes of these 15 distinct O antigen forms of L. Pneumophila were as target genes for multiplex PCR respectively, then primers and captured oligonucleotide probes were hybridizes with DNA microarray, which contained specific probes of Legionella pneumophila. Scanner was used to determinant the types of bacterium. The DNA microarray assay can detect 15 distinct O antigen forms of L. Pneumophila specially. The sensitivity of the DNA microarray was 10 ng. The detection method of 15 distinct O antigen forms of Legionella pneumophila by DNA microarray was established. The detection method has better specificity, sensitivity and repeatability.%建立一种多重PCR技术结合基因芯片检测方法,实现嗜肺军团菌15种血清型的快速、准确检测。根据嗜肺军团菌15种血清型的O抗原特异性基因设计并筛选合适的引物和探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有特异探针的芯片杂交。用扫描仪扫描,判定嗜肺军团菌的血清型。该基因芯片可特异性的检测嗜肺军团菌的15种血清型,具有良好的特异性,芯片纯菌DNA检测灵敏度为10 ng。所建立的嗜肺军团菌15种血清型基因芯片检测方法特异性好,灵敏度高,具有较好的实用性。

  5. Development of an RNA-based theophylline-specific microarray biosensor

    Jordan, Katherine M.

    We are developing an extremely sensitive and compact biosensor that is adaptable to a variety of target analytes. Hammerhead ribozymes have been engineered such that they rearrange from a catalytically inactive to an active conformation upon binding to a target molecule. A donor-acceptor fluorophore pair is coupled to the substrate RNA of such an aptamer, to form a complex referred to as an aptazyme, to monitor real-time cleavage activity in a fluid environment. The fluorophores interact by fluorescence resonance energy transfer (FRET) until binding of the target molecule, when the FRET signal breaks down as the substrate is cleaved and the products dissociate. FRET assays with immobilized aptazymes and using total internal reflection fluorescence (TIRF) microscopy on the single-molecule scale are presented showing an enhancement of substrate cleavage in the presence of theophylline over background. The aptazyme is hybridized onto a DNA microarray and incorporated into a chip specifically designed to allow for measurement in a controlled fluid environment. The use of these microarrays allows for either one spot, or a series of spots, to be addressed independently within the biosensor. This allows for multiple analytes to be tested simultaneously. An enhancement in the substrate cleavage is again observed in the presence of theophylline. Results are presented toward the characterization of a theophylline-specific aptamer-based biosensor using this RNA microarray platform and analogous measurement techniques.

  6. Adaptive response to DNA-damaging agents in natural Saccharomyces cerevisiae populations from "Evolution Canyon", Mt. Carmel, Israel.

    Gabriel A Lidzbarsky

    Full Text Available BACKGROUND: Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the "Evolution Canyon" microsite (Nahal Oren, Mt. Carmel, Israel. The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%-800% more on the "African" slope caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid "African" environment while the north-facing slope is represented by temperate, "European" forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS in order to evaluate the interslope effect on the strains' ability to withstand DNA-damaging agents. METHODOLOGY/PRINCIPAL FINDINGS: We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the "African" slope were more resilient to both UVA and MMS than the strains from the "European" slope. In contrast, we found that there was almost no difference between strains (with similar ploidy from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the "African" strains are more adapted to higher solar radiation than the "European" strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation. CONCLUSIONS/SIGNIFICANCE: Our results and the results of parallel studies with several other

  7. Influence of oxygen limitation, absence of the cytochrome bc(1) complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology.

    Hanke, Tanja; Richhardt, Janine; Polen, Tino; Sahm, Hermann; Bringer, Stephanie; Bott, Michael

    2012-02-10

    The genome-wide transcriptional responses of the strictly aerobic α-proteobacterium Gluconobacter oxydans 621H to oxygen limitation, to the absence of the cytochrome bc(1) complex, and to low pH were studied using DNA microarray analyses. Oxygen limitation caused expression changes of 486 genes, representing 20% of the chromosomal genes. Genes with an increased mRNA level included those for terminal oxidases, the cytochrome bc(1) complex, transhydrogenase, two alcohol dehydrogenases, heme biosynthesis, PTS proteins, proteins involved in cyclic diGMP synthesis and degradation, two sigma factors, flagella and chemotaxis proteins, several stress proteins, and a putative exporter protein. The downregulated genes comprised those for respiratory dehydrogenases, enzymes of central metabolism, PQQ biosynthesis, outer membrane receptors, Sec proteins, and proteins involved in transcription and translation. A ΔqrcABC mutant of G. oxydans showed a growth defect during cultivation on mannitol at pH 4 under oxygen saturation. Comparison of the transcriptomes of this mutant versus the wild type under these conditions revealed 51 differentially expressed genes. Interestingly, almost all of the 45 genes with increased expression in the ΔqrcABC mutant at pH 4 were also upregulated in the wild type grown at pH 6 under oxygen limitation. These results support an active role of the cytochrome bc(1) complex in G. oxydans respiration. The transcriptome comparison of G. oxydans wild type at pH 4 versus pH 6 in mannitol medium under oxygen-saturated conditions uncovered only 72 differentially expressed genes. The 35 upregulated genes included those for cytochrome bd oxidase, major polyol dehydrogenase, iron storage and oxidative stress proteins. Among the 37 downregulated genes were some encoding enzymes dealing with carbon dioxide, such as biotin carboxylase, biotin carboxyl carrier protein, and carboanhydrase. These results give first insights into global transcriptional responses of

  8. Application of DNA microarray in the identification of Candida spp. and mutations of ERG11 gene resulting in fluconazole resistance%DNA芯片鉴定念珠菌种和氟康唑耐药基因ERG11突变

    徐永豪; 王克玉; 李颖; 陈腊梅; 苏英; 孙青; 李春阳

    2009-01-01

    目的 建立DNA芯片技术鉴定念珠菌种和氟康唑耐药基因ERG11突变.方法 根据6种常见念珠菌内转录间隔(ITS2)区种特异性序列和白念珠菌ERG11基因中已证实可导致对氟康唑耐药的6种突变序列设计探针,制备DNA芯片,鉴定12条50 bp的念珠菌种特异性序列和ERG11突变序列及34株念珠菌(其中白念珠菌29株,热带念珠菌、光滑念珠菌、都柏林念珠菌、近平滑念珠菌和克柔念珠菌各1株).结果 ①芯片玤确鉴定12条人工合成序列;②正确鉴定34株试验菌株的菌种;③正确鉴定29株白念珠菌ERG11基因中可致耐药的已知突变.敏感性和特异性均为100%.结论 用DNA芯片进行念珠菌菌种鉴定和自念珠菌ERG11突变筛查,结果可靠.%Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains

  9. Navigating public microarray databases.

    Penkett, Christopher J; Bähler, Jürg

    2004-01-01

    With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources. PMID:18629145

  10. Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

    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.

  11. Population variability in biological adaptive responses to DNA damage and the shapes of carcinogen dose-response curves

    Carcinogen dose-response curves for both ionizing radiation and chemicals are typically assumed to be linear at environmentally relevant doses. This assumption is used to ensure protection of the public health in the absence of relevant dose-response data. A theoretical justification for the assumption has been provided by the argument that low dose linearity is expected when an exogenous agent adds to an ongoing endogenous process. Here, we use computational modeling to evaluate (1) how two biological adaptive processes, induction of DNA repair and cell cycle checkpoint control, may affect the shapes of dose-response curves for DNA-damaging carcinogens and (2) how the resulting dose-response behaviors may vary within a population. Each model incorporating an adaptive process was capable of generating not only monotonic dose-responses but also nonmonotonic (J-shaped) and threshold responses. Monte Carlo analysis suggested that all these dose-response behaviors could coexist within a population, as the spectrum of qualitative differences arose from quantitative changes in parameter values. While this analysis is largely theoretical, it suggests that (a) accurate prediction of the qualitative form of the dose-response requires a quantitative understanding of the mechanism (b) significant uncertainty is associated with human health risk prediction in the absence of such quantitative understanding and (c) a stronger experimental and regulatory focus on biological mechanisms and interindividual variability would allow flexibility in regulatory treatment of environmental carcinogens without compromising human health

  12. Cell-Based Microarrays for In Vitro Toxicology

    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.

  13. Radio-adaptive response using transcriptional status of DNA damage response genes in human peripheral blood lymphocytes

    Venous blood samples were collected from random healthy male individuals with informed consent. Peripheral Blood Mono Nuclear cells (PBMCs) were separated at 1 hour and 5 hours post-irradiation. Dose response was studied using 30 cGy, 60 cGy, 1.0 Gy and 2.0 Gy. For adaptive response study two priming doses (30 cGy and 60 cGy) was used followed by a challenging dose of 2.0 Gy after 4 hours. Total RNA was isolated and cDNA was synthesized. Relative quantitation was performed using a SYBR green based real time PCR with respect to beta actin. The results have shown significant up-regulation of DNA damage response genes like P53, GADD45A, CDKN1A and also in H2B, CTP Synthase and PLK3 at 5 hours post irradiation (P<0.001) as compared to 1 hour. In contrast, the transcriptional expression of ATM, ATR, MDM2, CDK2, cyclin E and cytokine genes remained same at both the time points (1 hour and 5 hours). Few genes like CDKN1A, GADD45A and P53 showed down regulation with 2 Gy at 5 hrs. Transcription profile at priming dose of 0.3 and 0.6 Gy followed by a challenging dose 2.0 Gy was studied. Adaptive response was observed at most of the DNA damage response genes. However, no difference was observed at master regulator ATM and P53. Similarly cytokines and histone modification gene also did not show any change. Detailed results will be discussed during the presentation

  14. Production of biomolecule microarrays through laser induced forward transfer

    Fernandez-Pradas, Juan Marcos; Serra, Pere; Colina, Monica; Morenza, Jose-Luis

    2004-10-01

    Biomolecule microarrays are a kind of biosensors that consist in patterns of different biological molecules immobilized on a solid substrate and capable to bind specifically to their complementary targets. In particular, DNA and protein microarrays have been revealed to be very efficient devices for genen and protein identification, what has converted them in powerful tools for many applications, like clinical diagnose, drug discovery analysis, genomics and proteomics. The production of these devices requires the manipulation of tiny amounts of a liquid solution containing biomolecules without damaging them. In this work laser induced forward transfer (LIFT) has been used for spotting a biomolecule in order to check the viability of this technique for the production of microarrays. A pulsed Nd:YAG laser beam (355 nm wavelength) has been used to transfer droplets of a biomolecule containing solution onto a solid slide. Optical microscopy of the transferred material has been carried out to investigate the morphological characteristics of the droplets obtained under different irradiation conditions. Afterwards, a DNA microarray has been spotted. The viability of the transference has been tested by checking the biological activity of the biomolecule in front of its specific complementary target. This has revealed that, indeed, the LIFT technique is adequate for the production of DNA microarrays.

  15. TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

    Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J

    2013-01-01

    Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenue...

  16. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    R. M. Farouk

    2014-09-01

    Full Text Available The complementary DNA (cDNA sequence considered the magic biometric technique for personal identification. Microarray image processing used for the concurrent genes identification. In this paper, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA microarray. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarray image resized and used as an input for the proposed artificial neural network. For matching and recognition, we have trained the artificial neural network. Recognition results are given for the galleries of cDNA sequences . The numerical results show that, the proposed matching technique is an effective in the cDNA sequences process. The experimental results of our matching approach using different databases shows that, the proposed technique is an effective matching performance.

  17. Symmetry-adapted digital modeling II. The double-helix B-DNA.

    Janner, A

    2016-05-01

    The positions of phosphorus in B-DNA have the remarkable property of occurring (in axial projection) at well defined points in the three-dimensional space of a projected five-dimensional decagonal lattice, subdividing according to the golden mean ratio τ:1:τ [with τ = (1+\\sqrt {5})/2] the edges of an enclosing decagon. The corresponding planar integral indices n1, n2, n3, n4 (which are lattice point coordinates) are extended to include the axial index n5 as well, defined for each P position of the double helix with respect to the single decagonal lattice ΛP(aP, cP) with aP = 2.222 Å and cP = 0.676 Å. A finer decagonal lattice Λ(a, c), with a = aP/6 and c = cP, together with a selection of lattice points for each nucleotide with a given indexed P position (so as to define a discrete set in three dimensions) permits the indexing of the atomic positions of the B-DNA d(AGTCAGTCAG) derived by M. J. P. van Dongen. This is done for both DNA strands and the single lattice Λ. Considered first is the sugar-phosphate subsystem, and then each nucleobase guanine, adenine, cytosine and thymine. One gets in this way a digital modeling of d(AGTCAGTCAG) in a one-to-one correspondence between atomic and indexed positions and a maximal deviation of about 0.6 Å (for the value of the lattice parameters given above). It is shown how to get a digital modeling of the B-DNA double helix for any given code. Finally, a short discussion indicates how this procedure can be extended to derive coarse-grained B-DNA models. An example is given with a reduction factor of about 2 in the number of atomic positions. A few remarks about the wider interest of this investigation and possible future developments conclude the paper. PMID:27126108

  18. CYR61 is a novel gene associated with temperature-dependent changes in fish metabolism as revealed by cDNA microarray analysis on a medaka Oryzias latipes cell line.

    Hirayama, Makoto; Ahsan, Md Nazmul; Mitani, Hiroshi; Watabe, Shugo

    2008-07-01

    A microarray comprising 3,514 cDNAs was constructed from a medaka EST library to elucidate the transcriptional responses associated with temperature shift from 25 to 15 degrees C in a medaka cell line. Microarray analysis revealed that the mRNA levels of 313 clones were significantly different in at least one combination of different incubation periods up to 7 days at a given incubation temperature or between 25 and 15 degrees C at a given incubation period (P poikilotherms. PMID:18286541

  19. Normalization Benefits Microarray-Based Classification

    Chen Yidong

    2006-01-01

    Full Text Available When using cDNA microarrays, normalization to correct labeling bias is a common preliminary step before further data analysis is applied, its objective being to reduce the variation between arrays. To date, assessment of the effectiveness of normalization has mainly been confined to the ability to detect differentially expressed genes. Since a major use of microarrays is the expression-based phenotype classification, it is important to evaluate microarray normalization procedures relative to classification. Using a model-based approach, we model the systemic-error process to generate synthetic gene-expression values with known ground truth. These synthetic expression values are subjected to typical normalization methods and passed through a set of classification rules, the objective being to carry out a systematic study of the effect of normalization on classification. Three normalization methods are considered: offset, linear regression, and Lowess regression. Seven classification rules are considered: 3-nearest neighbor, linear support vector machine, linear discriminant analysis, regular histogram, Gaussian kernel, perceptron, and multiple perceptron with majority voting. The results of the first three are presented in the paper, with the full results being given on a complementary website. The conclusion from the different experiment models considered in the study is that normalization can have a significant benefit for classification under difficult experimental conditions, with linear and Lowess regression slightly outperforming the offset method.

  20. Normalization Benefits Microarray-Based Classification

    Edward R. Dougherty

    2006-08-01

    Full Text Available When using cDNA microarrays, normalization to correct labeling bias is a common preliminary step before further data analysis is applied, its objective being to reduce the variation between arrays. To date, assessment of the effectiveness of normalization has mainly been confined to the ability to detect differentially expressed genes. Since a major use of microarrays is the expression-based phenotype classification, it is important to evaluate microarray normalization procedures relative to classification. Using a model-based approach, we model the systemic-error process to generate synthetic gene-expression values with known ground truth. These synthetic expression values are subjected to typical normalization methods and passed through a set of classification rules, the objective being to carry out a systematic study of the effect of normalization on classification. Three normalization methods are considered: offset, linear regression, and Lowess regression. Seven classification rules are considered: 3-nearest neighbor, linear support vector machine, linear discriminant analysis, regular histogram, Gaussian kernel, perceptron, and multiple perceptron with majority voting. The results of the first three are presented in the paper, with the full results being given on a complementary website. The conclusion from the different experiment models considered in the study is that normalization can have a significant benefit for classification under difficult experimental conditions, with linear and Lowess regression slightly outperforming the offset method.

  1. Low-density microarray technologies for rapid human norovirus genotyping

    Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, the use of low density DNA microarrays in conjuncti...

  2. Entwicklung und Vergleich biostatistischer Methoden zur Auswertung von Microarray Experimenten

    Clevert, Djork-Arné

    2012-01-01

    Motivation: Cost-effective oligonucleotide arrays like the Affymetrix SNP 6.0 and the Human Gene 1.1 ST are still the predominant technique to measure DNA copy number variations (CNVs) and gene expression, respectively. However, microarray data are characterized by high levels of noise induced by DNA preparation, staining, hybridization or measurement processes. This obscuring variation can blur out the signal of interest and, even worse, lead to spurious correlations which misguide the resea...

  3. A portable interferometric micro-array reader on image sensor

    Villar Zafra, Aitor

    2014-01-01

    [ANGLÈS] Microarrays constitute a valuable analytical tool for multiplex and high-throughput analysis and are widely used in genomics and proteomics with many potential applications. During the last decades, protein chips have found increasing acceptance for diagnostic applications due to several advantages over conventional bioanalysis such as miniaturization, parallelization, real-time and sensitivity. Even though the majority of DNA-sensor systems relies on labeling of DNA, the recent prog...

  4. Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins.

    Alex Boyko

    Full Text Available Epigenetic states and certain environmental responses in mammals and seed plants can persist in the next sexual generation. These transgenerational effects have potential adaptative significance as well as medical and agronomic ramifications. Recent evidence suggests that some abiotic and biotic stress responses of plants are transgenerational. For example, viral infection of tobacco plants and exposure of Arabidopsis thaliana plants to UVC and flagellin can induce transgenerational increases in homologous recombination frequency (HRF. Here we show that exposure of Arabidopsis plants to stresses, including salt, UVC, cold, heat and flood, resulted in a higher HRF, increased global genome methylation, and higher tolerance to stress in the untreated progeny. This transgenerational effect did not, however, persist in successive generations. Treatment of the progeny of stressed plants with 5-azacytidine was shown to decrease global genomic methylation and enhance stress tolerance. Dicer-like (DCL 2 and DCL3 encode Dicer activities important for small RNA-dependent gene silencing. Stress-induced HRF and DNA methylation were impaired in dcl2 and dcl3 deficiency mutants, while in dcl2 mutants, only stress-induced stress tolerance was impaired. Our results are consistent with the hypothesis that stress-induced transgenerational responses in Arabidopsis depend on altered DNA methylation and smRNA silencing pathways.

  5. Up-to-Date Applications of Microarrays and Their Way to Commercialization

    Sarah Schumacher

    2015-04-01

    Full Text Available This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

  6. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia.

    Bouquet, Fanny; Ousset, Marielle; Biard, Denis; Fallone, Frédérique; Dauvillier, Stéphanie; Frit, Philippe; Salles, Bernard; Muller, Catherine

    2011-06-01

    DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways. PMID:21576354

  7. Genetic Control or Repair and Adaptive Response to Low-Level DNA Damage

    J. E. Haber

    2009-10-05

    Research was focused on how a single double-strand break - a model of low-dose ionizing radiation-induced DNA damage - could be studied in a simple model system, budding yeast. Breaks were induced in several different ways. We used the site-specific HO endonuclease to create a single DSB in all cells of the population so that its fate could be extensively analyzed genetically and molecularly. We also used two heterologous systems, the plant DS element and the Rag1/Rag2 proteins, to generate different types of DSBs, these containing hairpin ends that needed to be cleaved open before end-joining could take place. All three approaches yielded important new findings. We also extended our analysis of the Mre11 protein that plays key roles in both NHEJ and in homologous recombination. Finally we analyzed the poorly understood recombination events that were independent of the key recombination protein, Rad52. This line of inquiry was strongly motivated by the fact that vertebrate cells do not rely strongly on Rad52 for homologous recombination, so that some clues about alternative mechanisms could be gained by understanding how Rad52-independent recombination occurred. We found that the Mre11 complex was the most important element in Rad52-independent recombination.

  8. Application of nanostructured biochips for efficient cell transfection microarrays

    Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  9. Evaluation of a DNA microarray (Check-MDR CT102) for rapid detection of TEM, SHV, and CTX-M extended-spectrum β-lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 carbapenemases.

    Naas, T.; Cuzon, G.; Bogaerts, P.; Glupczynski, Y.; Nordmann, P.

    2011-01-01

    The Check-MDR CT102 microarray, aimed at identifying bacteria producing extended-spectrum β-lactamase (ESBL) (SHV, TEM, and CTX-M) and carbapenemase (KPC, OXA-48, VIM, IMP, and NDM-1), was evaluated on a total of 144 Gram-negative strains expressing various β-lactamases. The sensitivity and specificity were 100% for most tested genes, suggesting that this assay allows accurate identification of common ESBL and carbapenemase producers from bacterial cultures.

  10. Microarray Scanner for Fluorescence Detection

    2003-01-01

    A novel pseudo confocal microarray scanner is introduced, in which one dimension scanning is performed by a galvanometer optical scanner and a telecentric objective, another dimension scanning is performed by a stepping motor.

  11. Microarrayed Materials for Stem Cells

    Ying Mei

    2012-01-01

    Stem cells hold remarkable promise for applications in disease modeling, cancer therapy, and regenerative medicine. Despite the significant progress made during the last decade, designing materials to control stem cell fate remains challenging. As an alternative, materials microarray technology has received great attention because it allows for high throughput materials synthesis and screening at a reasonable cost. Here, we discuss recent developments in materials microarray technology and th...

  12. Genomic interspecies microarray hybridization: rapid discovery of three thousand genes in the maize endophyte, Klebsiella pneumoniae 342, by microarray hybridization with Escherichia coli K-12 open reading frames.

    Dong, Y; Glasner, J D; Blattner, F R; Triplett, E W

    2001-04-01

    In an effort to efficiently discover genes in the diazotrophic endophyte of maize, Klebsiella pneumoniae 342, DNA from strain 342 was hybridized to a microarray containing 96% (n = 4,098) of the annotated open reading frames from Escherichia coli K-12. Using a criterion of 55% identity or greater, 3,000 (70%) of the E. coli K-12 open reading frames were also found to be present in strain 342. Approximately 24% (n = 1,030) of the E. coli K-12 open reading frames are absent in strain 342. For 1.6% (n = 68) of the open reading frames, the signal was too low to make a determination regarding the presence or absence of the gene. Genes with high identity between the two organisms are those involved in energy metabolism, amino acid metabolism, fatty acid metabolism, cofactor synthesis, cell division, DNA replication, transcription, translation, transport, and regulatory proteins. Functions that were less highly conserved included carbon compound metabolism, membrane proteins, structural proteins, putative transport proteins, cell processes such as adaptation and protection, and central intermediary metabolism. Open reading frames of E. coli K-12 with little or no identity in strain 342 included putative regulatory proteins, putative chaperones, surface structure proteins, mobility proteins, putative enzymes, hypothetical proteins, and proteins of unknown function, as well as genes presumed to have been acquired by lateral transfer from sources such as phage, plasmids, or transposons. The results were in agreement with the physiological properties of the two strains. Whole genome comparisons by genomic interspecies microarray hybridization are shown to rapidly identify thousands of genes in a previously uncharacterized bacterial genome provided that the genome of a close relative has been fully sequenced. This approach will become increasingly more useful as more full genome sequences become available. PMID:11282649

  13. [Research progress of probe design software of oligonucleotide microarrays].

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software. PMID:24804514

  14. Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes

    Eils Roland

    2005-11-01

    Full Text Available Abstract Background The extensive use of DNA microarray technology in the characterization of the cell transcriptome is leading to an ever increasing amount of microarray data from cancer studies. Although similar questions for the same type of cancer are addressed in these different studies, a comparative analysis of their results is hampered by the use of heterogeneous microarray platforms and analysis methods. Results In contrast to a meta-analysis approach where results of different studies are combined on an interpretative level, we investigate here how to directly integrate raw microarray data from different studies for the purpose of supervised classification analysis. We use median rank scores and quantile discretization to derive numerically comparable measures of gene expression from different platforms. These transformed data are then used for training of classifiers based on support vector machines. We apply this approach to six publicly available cancer microarray gene expression data sets, which consist of three pairs of studies, each examining the same type of cancer, i.e. breast cancer, prostate cancer or acute myeloid leukemia. For each pair, one study was performed by means of cDNA microarrays and the other by means of oligonucleotide microarrays. In each pair, high classification accuracies (> 85% were achieved with training and testing on data instances randomly chosen from both data sets in a cross-validation analysis. To exemplify the potential of this cross-platform classification analysis, we use two leukemia microarray data sets to show that important genes with regard to the biology of leukemia are selected in an integrated analysis, which are missed in either single-set analysis. Conclusion Cross-platform classification of multiple cancer microarray data sets yields discriminative gene expression signatures that are found and validated on a large number of microarray samples, generated by different laboratories and

  15. A genome-wide 20 K citrus microarray for gene expression analysis

    Gadea Jose

    2008-07-01

    Full Text Available Abstract Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database 1 was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global

  16. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    Ramirez, Lisa S; Wang, Jun

    2016-01-01

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

  17. A fisheye viewer for microarray-based gene expression data

    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.

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

    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.

  19. Laser-based patterning for transfected cell microarrays

    The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics.

  20. Laser-based patterning for transfected cell microarrays

    Hook, Andrew L; Creasey, Rhiannon; Voelcker, Nicolas H [Flinders University, GPO Box 2100, Bedford Park, SA 5042 (Australia); Hayes, Jason P [MiniFAB, 1 Dalmore Drive, Caribbean Park, Scoresby VIC 3179 (Australia); Thissen, Helmut, E-mail: Nico.Voelcker@flinders.edu.a [CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton VIC 3168 (Australia)

    2009-12-15

    The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics.

  1. DNA vaccines

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J.

    2013-01-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA...

  2. A methodology for global validation of microarray experiments

    Sladek Robert

    2006-07-01

    Full Text Available Abstract Background DNA microarrays are popular tools for measuring gene expression of biological samples. This ever increasing popularity is ensuring that a large number of microarray studies are conducted, many of which with data publicly available for mining by other investigators. Under most circumstances, validation of differential expression of genes is performed on a gene to gene basis. Thus, it is not possible to generalize validation results to the remaining majority of non-validated genes or to evaluate the overall quality of these studies. Results We present an approach for the global validation of DNA microarray experiments that will allow researchers to evaluate the general quality of their experiment and to extrapolate validation results of a subset of genes to the remaining non-validated genes. We illustrate why the popular strategy of selecting only the most differentially expressed genes for validation generally fails as a global validation strategy and propose random-stratified sampling as a better gene selection method. We also illustrate shortcomings of often-used validation indices such as overlap of significant effects and the correlation coefficient and recommend the concordance correlation coefficient (CCC as an alternative. Conclusion We provide recommendations that will enhance validity checks of microarray experiments while minimizing the need to run a large number of labour-intensive individual validation assays.

  3. The Stanford Tissue Microarray Database.

    Marinelli, Robert J; Montgomery, Kelli; Liu, Chih Long; Shah, Nigam H; Prapong, Wijan; Nitzberg, Michael; Zachariah, Zachariah K; Sherlock, Gavin J; Natkunam, Yasodha; West, Robert B; van de Rijn, Matt; Brown, Patrick O; Ball, Catherine A

    2008-01-01

    The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license. PMID:17989087

  4. Noise Removal From Microarray Images Using Maximum a Posteriori Based Bivariate Estimator

    A.Sharmila Agnal

    2013-01-01

    Full Text Available Microarray Image contains information about thousands of genes in an organism and these images are affected by several types of noises. They affect the circular edges of spots and thus degrade the image quality. Hence noise removal is the first step of cDNA microarray image analysis for obtaining gene expression level and identifying the infected cells. The Dual Tree Complex Wavelet Transform (DT-CWT is preferred for denoising microarray images due to its properties like improved directional selectivity and near shift-invariance. In this paper, bivariate estimators namely Linear Minimum Mean Squared Error (LMMSE and Maximum A Posteriori (MAP derived by applying DT-CWT are used for denoising microarray images. Experimental results show that MAP based denoising method outperforms existing denoising techniques for microarray images.

  5. Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways

    Thøgersen, Juliane Charlotte; Mørup, Morten; Pedersen, Søren Damkiær;

    2013-01-01

    introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first “Archetypal Analysis” of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas...... aeruginosa isolated from the airways of cystic fibrosis patients. RESULTS: Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene...... suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. CONCLUSIONS: Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering...

  6. Hyperspectral microarray scanning: impact on the accuracy and reliability of gene expression data

    Martinez M Juanita

    2005-05-01

    Full Text Available Abstract Background Commercial microarray scanners and software cannot distinguish between spectrally overlapping emission sources, and hence cannot accurately identify or correct for emissions not originating from the labeled cDNA. We employed our hyperspectral microarray scanner coupled with multivariate data analysis algorithms that independently identify and quantitate emissions from all sources to investigate three artifacts that reduce the accuracy and reliability of microarray data: skew toward the green channel, dye separation, and variable background emissions. Results Here we demonstrate that several common microarray artifacts resulted from the presence of emission sources other than the labeled cDNA that can dramatically alter the accuracy and reliability of the array data. The microarrays utilized in this study were representative of a wide cross-section of the microarrays currently employed in genomic research. These findings reinforce the need for careful attention to detail to recognize and subsequently eliminate or quantify the presence of extraneous emissions in microarray images. Conclusion Hyperspectral scanning together with multivariate analysis offers a unique and detailed understanding of the sources of microarray emissions after hybridization. This opportunity to simultaneously identify and quantitate contaminant and background emissions in microarrays markedly improves the reliability and accuracy of the data and permits a level of quality control of microarray emissions previously unachievable. Using these tools, we can not only quantify the extent and contribution of extraneous emission sources to the signal, but also determine the consequences of failing to account for them and gain the insight necessary to adjust preparation protocols to prevent such problems from occurring.

  7. Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis

    Bavykin, Sergei G.; Akowski, James P.; Zakhariev, Vladimir M.; Barsky, Viktor E.; Perov, Alexander N.; Mirzabekov, Andrei D.

    2001-01-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the ...

  8. StressDB: A Locally Installable Web-Based Relational Microarray Database Designed for Small User Communities

    Madhusmita Mitra; Nigam Shah; Lukas Mueller; Scuth Pin; Nina Fedoroff

    2002-01-01

    We have built a microarray database, StressDB, for management of microarray data from our studies on stress-modulated genes in Arabidopsis. StressDB provides small user groups with a locally installable web-based relational microarray database. It has a simple and intuitive architecture and has been designed for cDNA microarray technology users. StressDB uses Windows™ 2000 as the centralized database server with Oracle™ 8i as the relational database management system. It allows users to manag...

  9. Assessing the Application of Tissue Microarray Technology to Kidney Research

    Zhang, Ming-Zhi; Su, Yinghao; Yao, Bing; Zheng, Wei; deCaestecker, Mark; Harris, Raymond C.

    2010-01-01

    Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and...

  10. Robust Sequence Selection Method Used To Develop the FluChip Diagnostic Microarray for Influenza Virus

    Mehlmann, Martin; Dawson, Erica D.; Townsend, Michael B.; Smagala, James A.; Moore, Chad L.; Smith, Catherine B.; Cox, Nancy J.; Kuchta, Robert D.; Rowlen, Kathy L.

    2006-01-01

    DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to...

  11. A post-processing method for optimizing synthesis strategy for oligonucleotide microarrays

    Ning, Kang; Choi, Kwok Pui; Leong, Hon Wai; Zhang, Louxin

    2005-01-01

    The broad applicability of gene expression profiling to genomic analyses has generated huge demand for mass production of microarrays and hence for improving the cost effectiveness of microarray fabrication. We developed a post-processing method for deriving a good synthesis strategy. In this paper, we assessed all the known efficient methods and our post-processing method for reducing the number of synthesis cycles for manufacturing a DNA-chip of a given set of oligos. Our experimental resul...

  12. Microarray applications to understand the impact of exposure to environmental contaminants in wild dolphins (Tursiops truncatus).

    Mancia, Annalaura; Abelli, Luigi; Kucklick, John R; Rowles, Teresa K; Wells, Randall S; Balmer, Brian C; Hohn, Aleta A; Baatz, John E; Ryan, James C

    2015-02-01

    It is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. The health of an ecosystem is largely reflected in the health of its inhabitants. As an apex predator, the common bottlenose dolphin (Tursiops truncatus) can reflect the health of near shore marine ecosystems, and reflect coastal threats that pose risk to human health, such as legacy contaminants or marine toxins, e.g. polychlorinated biphenyls (PCBs) and brevetoxins. Major advances in the understanding of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of the development of cell-lines for use in in vitro experiments, the production of monoclonal antibodies to recognize dolphin proteins, the development of dolphin DNA microarrays to measure global gene expression and the sequencing of the dolphin genome. These advances may play a central role in understanding the complex and specialized biology of the dolphin with regard to how this species responds to an array of environmental insults. This work presents the creation, characterization and application of a new molecular tool to better understand the complex and unique biology of the common bottlenose dolphin and its response to environmental stress and infection. A dolphin oligo microarray representing 24,418 unigene sequences was developed and used to analyze blood samples collected from 69 dolphins during capture-release health assessments at five geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). The microarray was validated and tested for its ability to: 1) distinguish male from female dolphins; 2) differentiate dolphins inhabiting different geographic locations (Atlantic coasts vs the Gulf of Mexico); and 3) study in detail dolphins resident in one site, the Georgia coast, known to

  13. Adaptive differences in gene expression in European flounder ( Platichthys flesus )

    Larsen, Peter Foged; Eg Nielsen, Einar; Williams, T.D.;

    2007-01-01

    linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of...... differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low...... levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly...

  14. Microfluidic microarray systems and methods thereof

    West, Jay A. A.; Hukari, Kyle W.; Hux, Gary A.

    2009-04-28

    Disclosed are systems that include a manifold in fluid communication with a microfluidic chip having a microarray, an illuminator, and a detector in optical communication with the microarray. Methods for using these systems for biological detection are also disclosed.

  15. Microarray Developed on Plastic Substrates.

    Bañuls, María-José; Morais, Sergi B; Tortajada-Genaro, Luis A; Maquieira, Ángel

    2016-01-01

    There is a huge potential interest to use synthetic polymers as versatile solid supports for analytical microarraying. Chemical modification of polycarbonate (PC) for covalent immobilization of probes, micro-printing of protein or nucleic acid probes, development of indirect immunoassay, and development of hybridization protocols are described and discussed. PMID:26614067

  16. Improving comparability between microarray probe signals by thermodynamic intensity correction

    Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka; Willenbrock, Hanni; Nielsen, Henrik Bjørn

    different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities into...... determination of transcription start sites for a subset of yeast genes. In another application, we identify present/absent calls for probes hybridized to the sequenced Escherichia coli strain O157:H7 EDL933. The model improves the correct calls from 85 to 95% relative to raw intensity measures. The model thus...

  17. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Solanky, Dipesh; Shelley E Haydel

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicro...

  18. Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation

    Richard, Arianne C.; Lyons, Paul A.; Peters, James E.; Biasci, Daniele; Flint, Shaun M; James C Lee; McKinney, Eoin F; Siegel, Richard M.; Smith, Kenneth GC

    2014-01-01

    Background Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray u...

  19. Disc-based microarrays: principles and analytical applications.

    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays. PMID:26922341

  20. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    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)

  1. Chromosome microarrays in diagnostic testing: interpreting the genomic data.

    Peters, Greg B; Pertile, Mark D

    2014-01-01

    DNA-based Chromosome MicroArrays (CMAs) are now well established as diagnostic tools in clinical genetics laboratories. Over the last decade, the primary application of CMAs has been the genome-wide detection of a particular class of mutation known as copy number variants (CNVs). Since 2010, CMA testing has been recommended as a first-tier test for detection of CNVs associated with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies…in the post-natal setting. CNVs are now regarded as pathogenic in 14-18 % of patients referred for these (and related) disorders.Through consideration of clinical examples, and several microarray platforms, we attempt to provide an appreciation of microarray diagnostics, from the initial inspection of the microarray data, to the composing of the patient report. In CMA data interpretation, a major challenge comes from the high frequency of clinically irrelevant CNVs observed within "patient" and "normal" populations. As might be predicted, the more common and clinically insignificant CNVs tend to be the smaller ones resolution, and some miscalling of CNVs is unavoidable. In this, there is no ideal solution, but various strategies for handling noise are available. Even without solutions, consideration of these diagnostic problems per se is informative, as they afford critical insights into the biological and technical underpinnings of CNV discovery. These are indispensable to any clinician or scientist practising within the field of genome diagnostics. PMID:24870134

  2. Broad spectrum microarray for fingerprint-based bacterial species identification

    Frey Jürg E

    2010-02-01

    Full Text Available Abstract Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups.

  3. Exploiting fluorescence for multiplex immunoassays on protein microarrays

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

    2014-09-01

    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.

  4. Imaging combined autoimmune and infectious disease microarrays

    Ewart, Tom; Raha, Sandeep; Kus, Dorothy; Tarnopolsky, Mark

    2006-09-01

    Bacterial and viral pathogens are implicated in many severe autoimmune diseases, acting through such mechanisms as molecular mimicry, and superantigen activation of T-cells. For example, Helicobacter pylori, well known cause of stomach ulcers and cancers, is also identified in ischaemic heart disease (mimicry of heat shock protein 65), autoimmune pancreatitis, systemic sclerosis, autoimmune thyroiditis (HLA DRB1*0301 allele susceptibility), and Crohn's disease. Successful antibiotic eradication of H.pylori often accompanies their remission. Yet current diagnostic devices, and test-limiting cost containment, impede recognition of the linkage, delaying both diagnosis and therapeutic intervention until the chronic debilitating stage. We designed a 15 minute low cost 39 antigen microarray assay, combining autoimmune, viral and bacterial antigens1. This enables point-of-care serodiagnosis and cost-effective narrowly targeted concurrent antibiotic and monoclonal anti-T-cell and anti-cytokine immunotherapy. Arrays of 26 pathogen and 13 autoimmune antigens with IgG and IgM dilution series were printed in triplicate on epoxysilane covalent binding slides with Teflon well masks. Sera diluted 1:20 were incubated 10 minutes, washed off, anti-IgG-Cy3 (green) and anti-IgM-Dy647 (red) were incubated for 5 minutes, washed off and the slide was read in an ArrayWoRx(e) scanning CCD imager (Applied Precision, Issaquah, WA). As a preliminary model for the combined infectious disease-autoimmune diagnostic microarray we surveyed 98 unidentified, outdated sera that were discarded after Hepatitis B antibody testing. In these, significant IgG or IgM autoantibody levels were found: dsDNA 5, ssDNA 11, Ro 2, RNP 7, SSB 4, gliadin 2, thyroglobulin 13 cases. Since control sera showed no autoantibodies, the high frequency of anti-DNA and anti-thyroglobulin antibodies found in infected sera lend increased support for linkage of infection to subsequent autoimmune disease. Expansion of the antigen

  5. Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus

    Gresham, D.; Usaite, Renata; Germann, S.M.;

    2010-01-01

    To study adaptive evolution in defined environments, we performed evolution experiments with Saccharomyces cerevisiae (yeast) in nitrogen-limited chemostat cultures. We used DNA microarrays to identify copy-number variation associated with adaptation and observed frequent amplifications and...... chromosomal locus. The GAP1circle is selected following prolonged culturing in L-glutamine-limited chemostats in a manner analogous to the selection of oncogenes present on double minutes in human cancers. Clones carrying only the gap1 Delta allele were selected under various non-amino acid nitrogen...

  6. Microarray analysis in pulmonary hypertension.

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea; Kwapiszewska, Grazyna

    2016-07-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  7. Optimisation algorithms for microarray biclustering.

    Perrin, Dimitri; Duhamel, Christophe

    2013-01-01

    In providing simultaneous information on expression profiles for thousands of genes, microarray technologies have, in recent years, been largely used to investigate mechanisms of gene expression. Clustering and classification of such data can, indeed, highlight patterns and provide insight on biological processes. A common approach is to consider genes and samples of microarray datasets as nodes in a bipartite graphs, where edges are weighted e.g. based on the expression levels. In this paper, using a previously-evaluated weighting scheme, we focus on search algorithms and evaluate, in the context of biclustering, several variations of Genetic Algorithms. We also introduce a new heuristic "Propagate", which consists in recursively evaluating neighbour solutions with one more or one less active conditions. The results obtained on three well-known datasets show that, for a given weighting scheme, optimal or near-optimal solutions can be identified. PMID:24109756

  8. How Can Microarrays Unlock Asthma?

    Alen Faiz

    2012-01-01

    Full Text Available Asthma is a complex disease regulated by the interplay of a large number of underlying mechanisms which contribute to the overall pathology. Despite various breakthroughs identifying genes related to asthma, our understanding of the importance of the genetic background remains limited. Although current therapies for asthma are relatively effective, subpopulations of asthmatics do not respond to these regimens. By unlocking the role of these underlying mechanisms, a source of novel and more effective treatments may be identified. In the new age of high-throughput technologies, gene-expression microarrays provide a quick and effective method of identifying novel genes and pathways, which would be impossible to discover using an individual gene screening approach. In this review we follow the history of expression microarray technologies and describe their contributions to advancing our current knowledge and understanding of asthma pathology.

  9. Lipid Microarray Biosensor for Biotoxin Detection.

    Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.; Edel, Joshua B.; Meyer, Grant D.; Craighead, Harold G.

    2006-05-01

    We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates by TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4

  10. Seeded Bayesian Networks: Constructing genetic networks from microarray data

    Quackenbush John

    2008-07-01

    Full Text Available Abstract Background DNA microarrays and other genomics-inspired technologies provide large datasets that often include hidden patterns of correlation between genes reflecting the complex processes that underlie cellular metabolism and physiology. The challenge in analyzing large-scale expression data has been to extract biologically meaningful inferences regarding these processes – often represented as networks – in an environment where the datasets are often imperfect and biological noise can obscure the actual signal. Although many techniques have been developed in an attempt to address these issues, to date their ability to extract meaningful and predictive network relationships has been limited. Here we describe a method that draws on prior information about gene-gene interactions to infer biologically relevant pathways from microarray data. Our approach consists of using preliminary networks derived from the literature and/or protein-protein interaction data as seeds for a Bayesian network analysis of microarray results. Results Through a bootstrap analysis of gene expression data derived from a number of leukemia studies, we demonstrate that seeded Bayesian Networks have the ability to identify high-confidence gene-gene interactions which can then be validated by comparison to other sources of pathway data. Conclusion The use of network seeds greatly improves the ability of Bayesian Network analysis to learn gene interaction networks from gene expression data. We demonstrate that the use of seeds derived from the biomedical literature or high-throughput protein-protein interaction data, or the combination, provides improvement over a standard Bayesian Network analysis, allowing networks involving dynamic processes to be deduced from the static snapshots of biological systems that represent the most common source of microarray data. Software implementing these methods has been included in the widely used TM4 microarray analysis package.

  11. Fast protocol for extraction of DNA from Prosopis spp leaves (plant adapted to arid environment) without liquid nitrogen.

    Michel-López, C Y; González-Mendoza, D; Grimaldo-Juarez, O

    2013-01-01

    The extraction of high-quality genomic DNA from Prosopis spp for polymerase chain reaction (PCR) amplification is complicated, owing to the presence of a high percentage of secondary metabolites that bind to or co-precipitate with nucleic acids. In the present study, we report a modified sodium dodecyl sulfate/phenol protocol that eliminates the use of liquid nitrogen in the maceration process, β-mercaptoethanol in the buffer extraction, and the ethanol precipitation step. The A₂₆₀/A₂₈₀ absorbance ratios of the isolated DNA were approximately 2.0 to 1.9, suggesting that the DNA fraction was pure and can be used for further PCR analysis. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. Finally, this proposal represents an alternative fast, cheap, and effective method for the isolation of genomic DNA from fresh leaves of Prosopis spp, even in low-technology laboratories. PMID:24089098

  12. Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

    Shepard, Jason R. E.

    2009-05-01

    The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

  13. DNA microarray profiling of a diverse collection of nosocomial methicillin-resistant staphylococcus aureus isolates assigns the majority to the correct sequence type and staphylococcal cassette chromosome mec (SCCmec) type and results in the subsequent identification and characterization of novel SCCmec-SCCM1 composite islands.

    Shore, Anna C

    2012-10-01

    One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100\\/107) were assigned an ST, with 98% (98\\/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec\\/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50\\/fusC. Novel SCCmec\\/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCC(M1) from ST8\\/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100\\/107) and immune evasion cluster (91%; 97\\/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs\\/STs and SCCmec types and provided further evidence of the diversity of SCCmec\\/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate.

  14. Classification analysis of microarray data based on ontological engineering

    LI Guo-qi; SHENG Huan-ye

    2007-01-01

    Background knowledge is important for data mining, especially in complicated situation. Ontological engineering is the successor of knowledge engineering. The sharable knowledge bases built on ontology can be used to provide background knowledge to direct the process of data mining. This paper gives a common introduction to the method and presents a practical analysis example using SVM (support vector machine) as the classifier. Gene Ontology and the accompanying annotations compose a big knowledge base, on which many researches have been carried out. Microarray dataset is the output of DNA chip.With the help of Gene Ontology we present a more elaborate analysis on microarray data than former researchers. The method can also be used in other fields with similar scenario.

  15. Prolonged durability of electroporation microarrays as a result of addition of saccharides to nucleic acids.

    Fujimoto, Hiroyuki; Kato, Koichi; Iwata, Hiroo

    2009-01-01

    The electroporation microarray is a useful tool for high-throughput analysis of gene functions. However, transfection efficiency is greatly impaired by storage of the microarrays, due to water evaporation from arrayed nucleotides. In this study, we aimed at evaluating the effect of saccharides and sugar alcohols, added to the solution of the plasmid DNA or small interfering RNA (siRNA). Microarrays loaded with plasmids and siRNAs were prepared with various polyols including sugars and sugar alcohols. After storage of these microarrays at different temperatures for various time periods, transfection efficiency was evaluated using human embryonic kidney cells. In the case of plasmid-loaded microarrays, addition of monosaccharides (glucose, fructose), disaccharides (trehalose, sucrose), and trisaccharide (raffinose) served to retain transfection efficiency at a reasonably high level after storage at -20 degrees C. The observed effects may be because moisture retention serves to maintain the solubility of DNA. In contrast, polysaccharide (dextran) and sugar alcohol (glycerol) had insignificant effects on retention of transfection efficiency. On the other hand, addition of saccharides and sugar alcohols had insignificant effects on the transfection of siRNA after storage of a microarray at 25 degrees C for 7 days, presumably due to the intrinsically-high solubility of siRNA which consists of short nucleotides. PMID:18989662

  16. Interpreting Microarray Data to Build Models of Microbial Genetic Regulation Networks

    Sokhansanj, B; Garnham, J B; Fitch, J P

    2002-01-23

    Microarrays and DNA chips are an efficient, high-throughput technology for measuring temporal changes in the expression of message RNA (mRNA) from thousands of genes (often the entire genome of an organism) in a single experiment. A crucial drawback of microarray experiments is that results are inherently qualitative: data are generally neither quantitatively repeatable, nor may microarray spot intensities be calibrated to in vivo mRNA concentrations. Nevertheless, microarrays represent by the far the cheapest and fastest way to obtain information about a cells global genetic regulatory networks. Besides poor signal characteristics, the massive number of data produced by microarray experiments poses challenges for visualization, interpretation and model building. Towards initial model development, we have developed a Java tool for visualizing the spatial organization of gene expression in bacteria. We are also developing an approach to inferring and testing qualitative fuzzy logic models of gene regulation using microarray data. Because we are developing and testing qualitative hypotheses that do not require quantitative precision, our statistical evaluation of experimental data is limited to checking for validity and consistency. Our goals are to maximize the impact of inexpensive microarray technology, bearing in mind that biological models and hypotheses are typically qualitative.

  17. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  18. Enhancing the quality metric of protein microarray image

    王立强; 倪旭翔; 陆祖康; 郑旭峰; 李映笙

    2004-01-01

    The novel method of improving the quality metric of protein microarray image presented in this paper reduces impulse noise by using an adaptive median filter that employs the switching scheme based on local statistics characters; and achieves the impulse detection by using the difference between the standard deviation of the pixels within the filter window and the current pixel of concern. It also uses a top-hat filter to correct the background variation. In order to decrease time consumption, the top-hat filter core is cross structure. The experimental results showed that, for a protein microarray image contaminated by impulse noise and with slow background variation, the new method can significantly increase the signal-to-noise ratio, correct the trends in the background, and enhance the flatness of the background and the consistency of the signal intensity.

  19. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    Medrano Juan F

    2006-03-01

    Full Text Available Abstract Background The increasing use of DNA microarrays for genetical genomics studies generates a need for platforms with complete coverage of the genome. We have compared the effective gene coverage in the mouse genome of different commercial and noncommercial oligonucleotide microarray platforms by performing an in-house gene annotation of probes. We only used information about probes that is available from vendors and followed a process that any researcher may take to find the gene targeted by a given probe. In order to make consistent comparisons between platforms, probes in each microarray were annotated with an Entrez Gene id and the chromosomal position for each gene was obtained from the UCSC Genome Browser Database. Gene coverage was estimated as the percentage of Entrez Genes with a unique position in the UCSC Genome database that is tested by a given microarray platform. Results A MySQL relational database was created to store the mapping information for 25,416 mouse genes and for the probes in five microarray platforms (gene coverage level in parenthesis: Affymetrix430 2.0 (75.6%, ABI Genome Survey (81.24%, Agilent (79.33%, Codelink (78.09%, Sentrix (90.47%; and four array-ready oligosets: Sigma (47.95%, Operon v.3 (69.89%, Operon v.4 (84.03%, and MEEBO (84.03%. The differences in coverage between platforms were highly conserved across chromosomes. Differences in the number of redundant and unspecific probes were also found among arrays. The database can be queried to compare specific genomic regions using a web interface. The software used to create, update and query the database is freely available as a toolbox named ArrayGene. Conclusion The software developed here allows researchers to create updated custom databases by using public or proprietary information on genes for any organisms. ArrayGene allows easy comparisons of gene coverage between microarray platforms for any region of the genome. The comparison presented here

  20. Genes transactivated by hepatitis C virus core protein, a microarray assay

    Min Liu; Shu-Lin Zhang; Jun Cheng; Yan Liu; Lin Wang; Qing Shao; Jian Zhang; Shu-Mei Lin

    2005-01-01

    AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection.METHODS: Reverse transcribed cDNA was subjected tomicroarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods.RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene namedHCTP4 was cloned with molecular biological method in combination with bioinformatics method.CONCLUSION: HCV core is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.

  1. High quality protein microarray using in situ protein purification

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  2. Study on Wusan Granule Anti-tumor Related Target Gene Screened by Cdna Microarray

    YOU Zi-li; SHI Jin-ping; CHEN Hai-hong

    2006-01-01

    To screen Wusan Granule anti-tumor related target gene using cDNA microarray technique, both mRNA from Lewis lung carcinoma tissues treated by Wusan Granule and untreated control are reversibly transcribed to prepare cDNA probes which are labeled by Cy5 and Cy3. Then, the probes are hybridized to the mice cDNA microarray type MGEC-20S. After hybridization, the cDNA microarray is scanned by ScanArray 3 000 scanner and the data is analyzed by ImaGene 3 software to screen the differentially expressed genes. There are 45 differentially expressed genes including 18 known genes and 27 unknown genes between the two groups, and among them, 20 elevated genes and 25 reduced genes are identified. Additionally, the genes related to invasion and metastasis of malignant carcinomas are down-regulated and the genes related to apoptosis are up-regulated. The cDNA microarray technique is a high-throughput approach to screen the Wusan Granule anti-tumor related target genes, which allow us to explore the molecular biological mechanism on a genomic scale.

  3. Construction of metastatic spinal cancer tissue microarrays

    Yang Xinghai; Chen Huajiang; Xiao Jianru; Yuan Wen; Jia Lianshun

    2009-01-01

    Objective: To explore the construction of metastatic spinal cancer (MSC) tissue microarrays and validate its value in immunohistochemical study of MSC. Methods: Paraffin-embedded specimens from 71 MSC cases and 6 primary tumor cases were selected as donor blocks and prepared into MSC tissue microarrays by tissue array arrangement, the steps of which included location, punching, sampling, sample seeding, and re-diagnosis by hematoxylin-eosin (HE) as well as MMP-9 and MMP-14 immunohistochemical staining. Results: The MSC tissue microarrays thus constructed were intact and crackless, containing 154 complete and well arranged microarray points. None of the sectioned tissue microarrays was lost, and the results of HE staining was consistent with the primary pathologic diagnoses. Immunohistochemical staining was also good without non-specific or marginal effect. Conclusion: The MSC tissue microarrays have a high value in the immunohistochemical study of MSC.

  4. Unraveling the Rat Intestine, Spleen and Liver Genome-Wide Transcriptome after the Oral Administration of Lavender Oil by a Two-Color Dye-Swap DNA Microarray Approach.

    Hiroko Kubo

    Full Text Available The use of lavender oil (LO--a commonly, used oil in aromatherapy, with well-defined volatile components linalool and linalyl acetate--in non-traditional medicine is increasing globally. To understand and demonstrate the potential positive effects of LO on the body, we have established an animal model in this current study, investigating the orally administered LO effects genome wide in the rat small intestine, spleen, and liver. The rats were administered LO at 5 mg/kg (usual therapeutic dose in humans followed by the screening of differentially expressed genes in the tissues, using a 4×44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA in conjunction with a dye-swap approach, a novelty of this study. Fourteen days after LO treatment and compared with a control group (sham, a total of 156 and 154 up (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes, 174 and 66 up- (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes, and 222 and 322 up- (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes showed differential expression at the mRNA level in the small intestine, spleen and liver, respectively. The reverse transcription-polymerase chain reaction (RT-PCR validation of highly up- and down-regulated genes confirmed the regulation of the Papd4, Lrp1b, Alb, Cyr61, Cyp2c, and Cxcl1 genes by LO as examples in these tissues. Using bioinformatics, including Ingenuity Pathway Analysis (IPA, differentially expressed genes were functionally categorized by their Gene Ontology (GO and biological function and network analysis, revealing their diverse functions and potential roles in LO-mediated effects in rat. Further IPA analysis in particular unraveled the presence of novel genes, such as Papd4, Or8k5, Gprc5b, Taar5, Trpc6, Pld2 and Onecut3 (up-regulated top molecules and Tnf, Slc45a4, Slc25a23 and Samt4 (down-regulated top molecules, to be influenced by LO treatment in the small intestine, spleen and

  5. Unraveling the Rat Intestine, Spleen and Liver Genome-Wide Transcriptome after the Oral Administration of Lavender Oil by a Two-Color Dye-Swap DNA Microarray Approach.

    Kubo, Hiroko; Shibato, Junko; Saito, Tomomi; Ogawa, Tetsuo; Rakwal, Randeep; Shioda, Seiji

    2015-01-01

    The use of lavender oil (LO)--a commonly, used oil in aromatherapy, with well-defined volatile components linalool and linalyl acetate--in non-traditional medicine is increasing globally. To understand and demonstrate the potential positive effects of LO on the body, we have established an animal model in this current study, investigating the orally administered LO effects genome wide in the rat small intestine, spleen, and liver. The rats were administered LO at 5 mg/kg (usual therapeutic dose in humans) followed by the screening of differentially expressed genes in the tissues, using a 4×44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a dye-swap approach, a novelty of this study. Fourteen days after LO treatment and compared with a control group (sham), a total of 156 and 154 up (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, 174 and 66 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, and 222 and 322 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes showed differential expression at the mRNA level in the small intestine, spleen and liver, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) validation of highly up- and down-regulated genes confirmed the regulation of the Papd4, Lrp1b, Alb, Cyr61, Cyp2c, and Cxcl1 genes by LO as examples in these tissues. Using bioinformatics, including Ingenuity Pathway Analysis (IPA), differentially expressed genes were functionally categorized by their Gene Ontology (GO) and biological function and network analysis, revealing their diverse functions and potential roles in LO-mediated effects in rat. Further IPA analysis in particular unraveled the presence of novel genes, such as Papd4, Or8k5, Gprc5b, Taar5, Trpc6, Pld2 and Onecut3 (up-regulated top molecules) and Tnf, Slc45a4, Slc25a23 and Samt4 (down-regulated top molecules), to be influenced by LO treatment in the small intestine, spleen and liver

  6. Microgeographical population structure and adaptation in Atlantic cod Gadus morhua: spatio-temporal insights from gene-associated DNA markers

    Poulsen, Nina Aagaard; Hemmer-Hansen, Jakob; Loeschcke, V.; Carvalho, G.R.; Eg Nielsen, Einar

    2011-01-01

    populations, indicating long-term temporal adaptive stability driven by strong local selection. In an environmentally dynamic area, on the other hand, patterns of genetic structuring were more variable. Overall, our results not only suggest separation of populations under both evolutionary and ecological......Recent technical advances have stimulated studies on spatial scales of adaptive genetic variation in marine fishes. However, very few studies have combined spatial and temporal sampling to investigate adaptive genetic structuring at local and microgeographical scales, i.e. scales at which neutral...... stability, we included long- and short-term (i.e. from 24 to 38 and from 8 to 11 yr, ­respectively) temporally replicated samples from a subset of populations. As expected, we found very low levels of neutral genetic population structure (FST = 0.003). Three specific loci, however, showed highly elevated...

  7. MICROARRAYS AND THEIR POTENTIAL IN MEDICINE

    Erick Ling; Jie Xu

    2003-01-01

    Advancement in microarray technology can revolutionize many aspects of medicine. Microarrays have applications in gene expression profiling, genotyping, mutation analysis, gene identification, and pharmacology. This paper provides a brief review on the use of microarrays in studies of cancer, infectious diseases, chromosome disorders, neurological/mental disorders, and drugs, along with a prospect on its great potential in diagnosis, prognosis and the treatment of human diseases.

  8. Comprehensive comparison of six microarray technologies

    Yauk, Carole L.; Berndt, M. Lynn; Williams, Andrew; Douglas, George R

    2004-01-01

    Microarray technology is extensively used in biological research. The applied technologies vary greatly between laboratories, and outstanding questions remain regarding the degree of correlation among approaches. Recently, there has been a drive toward ensuring high-quality microarray data by the implementation of MIAME (Minimal Information About a Microarray Experiment) guidelines and an emphasis on ensuring public-availability to all datasets. However, despite its current widespread use and...

  9. Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI-TOF Mass Spectrometry

    Deyu Li

    2010-01-01

    Full Text Available DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N 6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol of 3,N 4-ethenocytosine are reported for the first time. It is also demonstrated that O 6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro.

  10. Comparison of vaccines for induction of heterosubtypic immunity to influenza A virus: cold-adapted vaccine versus DNA prime-adenovirus boost strategies.

    Lo, Chia-Yun; Wu, Zhengqi; Misplon, Julia A; Price, Graeme E; Pappas, Claudia; Kong, Wing-Pui; Tumpey, Terrence M; Epstein, Suzanne L

    2008-04-16

    Influenza epidemics or pandemics can arise for which strain- or subtype-matched vaccines are unavailable. Heterosubtypic immunity (Het-I) targeting conserved influenza A antigens could reduce morbidity and mortality during preparation of matched vaccines. Various vaccines inducing Het-I in animals have been studied separately using different viruses and conditions, but effectiveness for inducing Het-I has not been directly compared. The present studies compared immunization with cold-adapted (ca) viruses to DNA prime-recombinant adenovirus (rAd) boost vaccination to conserved antigens nucleoprotein (NP), matrix-2 (M2), or A/NP+M2. Both ca and DNA-rAd vaccinations induced antibody and T cell responses, and protected against lethal H1N1 challenge. Only A/NP+M2 DNA-rAd protected against challenge with highly pathogenic A/Vietnam/1203/2004 (H5N1); ca vaccine did not. Existing ca vaccines may provide some Het-I, but experimental vaccination focusing on conserved antigens was more effective in this model for protection against a divergent, highly pathogenic virus. PMID:18378366

  11. A theoretical analysis of codon adaptation index of the Boophilus microplus bm86 gene directed to the optimization of a DNA vaccine.

    Ruiz, Lina María; Armengol, Gemma; Habeych, Edwin; Orduz, Sergio

    2006-04-21

    DNA vaccines utilize host cell molecules for gene transcription and translation to proteins, and the interspecific difference of codon usage is one of the major obstacles for effective induction of specific and strong immune response. In an attempt to improve codon usage effects of DNA vaccine on protein expression, a quantitative study was conducted to clarify the relationship of codon usage in the tick gene bm86 and its potential expression in bovine cells. The calculated relative synonymous codon usage (RSCU) and codon adaptation index (CAI) values of bm86 from Boophilus microplus and a set of 14 highly expressed genes from Bos taurus indicated that some codons utilized frequently in bm86 are rarely used in B. taurus genes and vice versa. The different translational efficiencies obtained suggested that after DNA vaccination using the wild bm86 gene, the protein Bm86 would be expressed in bovines, but it would not be the optimum sequence. However, using the codon-optimized bm86 gene to bovines, whose sequence was theoretically designed, would probably improve the level of the immune response generated against ticks. PMID:16171828

  12. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    R. M. Farouk

    2014-07-01

    Full Text Available The complementary DNA (cDNA sequence considered th e magic biometric technique for personal identification. Microarray image processing used fo r the concurrent genes identification. In this pape r, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA micro array. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarr ay image resized and used as an input for the proposed artificial neural network. For matching an d recognition, we have trained the artificial neura l network. Recognition results are given for the gall eries of cDNA sequences . The numerical results sho w that, the proposed matching technique is an effecti ve in the cDNA sequences process. The experimental results of our matching approach using different da tabases shows that, the proposed technique is an effective matching performance.

  13. A reliable method to concentrate circulating DNA.

    Bryzgunova, Olga; Bondar, Anna; Morozkin, Evgeniy; Mileyko, Vladislav; Vlassov, Valentin; Laktionov, Pavel

    2011-01-15

    Concentration of circulating DNA probes is required to increase the amount of DNA involved in subsequent study (by polymerase chain reaction, sequencing, and microarray). This work was dedicated to the comparison of five different methods used for concentration of DNA circulating in blood. Precipitation of circulating DNA with acetone in the presence of triethylamine provides minimal DNA loss, high reproducibility, and at least three times higher DNA yield in comparison with the standard ethanol protocol. PMID:20828533

  14. Microarray-based genotyping of Salmonella: Inter-laboratory evaluation of reproducibility and standardization potential

    Grønlund, Hugo Ahlm; Riber, Leise; Vigre, Håkan;

    2011-01-01

    Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variationamong different isolates in order to control pathogen-induced infections. Microarray...... technology is a promising diagnostic tool that provides genomic information onmany genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was...... designed inwhich the agreement of data fromaDNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of...

  15. SNP typing on the NanoChip electronic microarray

    Børsting, Claus; Sanchez Sanchez, Juan Jose; Morling, Niels

    2005-01-01

    We describe a single nucleotide polymorphism (SNP) typing protocol developed for the NanoChip electronic microarray. The NanoChip array consists of 100 electrodes covered by a thin hydrogel layer containing streptavidin. An electric currency can be applied to one, several, or all electrodes at the...... the bound DNA. Base stacking between the short reporter and the longer stabilizer oligo stabilizes the binding of a matching reporter, whereas the binding of a reporter carrying a mismatch in the SNP position will be relatively weak. Thermal stringency is applied to the NanoChip array according to a...

  16. 2型登革病毒检测基因芯片的研制%Construction of a Diagnostic cDNA Microarray for Serotype 2 Dengue Virus Detection

    肖维威; 马文丽; 王洪敏; 黄海; 王艳; 张宝; 郑文岭

    2003-01-01

    利用长片断PCR扩增含几乎全长的2型登革病毒cDNA,酶切PCR扩增产物,通过建立2型登革病毒cDNA文库获取芯片探针.用点样仪将探针制备成2型登革病毒检测基因芯片.杂交时采用限制性显示(Restriction Display RD)技术标记样品,Scan Array芯片扫描仪检测杂交信号.杂交结果显示,样品和2型登革病毒基因芯片杂交的敏感性强、特异性高.

  17. Development of gene microarray in screening differently expressed genes in keloid and normal-control skin

    陈伟; 付小兵; 葛世丽; 孙晓庆; 周岗; 赵志力; 盛志勇

    2004-01-01

    Background Keloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins. Methods The polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs, with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-β1 (TGF-β1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray. Results Among the 8400 human genes, 402 were detected with different expression levels between keloid and normal control skins. Two hundred and fifty genes, including TGF-β1 and c-myc, were up-regulated and 152 genes were down-regulated. Higher expressions of TGF-β1 and c-myc in keloid were also revealed using RT-PCR and Northern blot methods. Conclusion cDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genes involved.

  18. Microarray, SAGE and their applications to cardiovascular diseases

    2002-01-01

    The wealth of DNA data generated by the human genome project coupling with recently invented high-throughput gene expression profiling techniques has dramatically sped up the process for biomedical researchers on elucidating the role of genes in human diseases. One powerful method to reveal insight into gene functions is the systematic analysis of gene expression. Two popular high-throughput gene expression technologies, microarray and Serial Analysis of Gene Expression (SAGE) are capable of producing large amounts of gene expression data with the potential of providing novel insights into fundamental disease processes, especially complex syndromes such as cardiovascular disease, whose etiologies are due to multiple genetic factors and their interplay with the environment. Microarray and SAGE have already been used to examine gene expression patterns of cell-culture, animal and human tissues models of cardiovascular diseases. In this review, we will first give a brief introduction of microarray and SAGE technologies and point out their limitations. We will then discuss the major discoveries and the new biological insightsthat have emerged from their applications to cardiovascular diseases. Finally we will touch upon potential challenges and future developments in this area.

  19. Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

    Francois, Patrice; Charbonnier, Yvan; Jacquet, Jean; Utinger, Dominic; Bento, Manuela; Lew, Daniel; Kresbach, Gerhard M; Ehrat, Markus; Schlegel, Werner; Schrenzel, Jacques

    2006-06-01

    Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription. PMID:16216356

  20. Retrieving relevant experiments: The case of microRNA microarrays.

    Açıcı, Koray; Terzi, Yunus Kasım; Oğul, Hasan

    2015-08-01

    Content-based retrieval of biological experiments in large public repositories is a recent challenge in computational biology and bioinformatics. The task is, in general, to search in a database using a query-by-example without any experimental meta-data annotation. Here, we consider a more specific problem that seeks a solution for retrieving relevant microRNA experiments from microarray repositories. A computational framework is proposed with this objective. The framework adapts a normal-uniform mixture model for identifying differentially expressed microRNAs in microarray profiling experiments. A rank-based thresholding scheme is offered to binarize real-valued experiment fingerprints based on differential expression. An effective similarity metric is introduced to compare categorical fingerprints, which in turn infers the relevance between two experiments. Two different views of experimental relevance are evaluated, one for disease association and another for embryonic germ layer, to discern the retrieval ability of the proposed model. To the best of our knowledge, the experiment retrieval task is investigated for the first time in the context of microRNA microarrays. PMID:26116091

  1. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

    Gasiunas, Giedrius; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2012-01-01

    Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system in...

  2. Understanding mechanisms of vitiligo development in Smyth line of chickens by transcriptomic microarray analysis of evolving autoimmune lesions

    Shi Fengying

    2012-04-01

    Full Text Available Abstract Background The Smyth line (SL of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather of SL chickens at different vitiligo (SLV states. Results Compared to the reference sample, which was from Brown line chickens (the parental control, 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE (P ≤ 0.05 in feather samples collected from SL chickens that never developed SLV (NV, from SLV chickens prior to SLV onset (EV, during active loss of pigmentation (AV, and after complete loss of melanocytes (CV. Comparisons of gene expression levels within SL samples (NV, EV, AV and CV revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death. Conclusions The microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune

  3. Diagnostic Yield of Chromosomal Microarray Analysis in an Autism Primary Care Practice: Which Guidelines to Implement?

    McGrew, Susan G.; Peters, Brittany R.; Crittendon, Julie A.; Veenstra-VanderWeele, Jeremy

    2012-01-01

    Genetic testing is recommended for patients with ASD; however specific recommendations vary by specialty. American Academy of Pediatrics and American Academy of Neurology guidelines recommend G-banded karyotype and Fragile X DNA. The American College of Medical Genetics recommends Chromosomal Microarray Analysis (CMA). We determined the yield of…

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

    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...

  5. A Bifidobacterium mixed-species microarray for high resolution discrimination between intestinal bifidobacteria

    Boesten, R.J.; Schuren, F.H.; Vos, de W.M.

    2009-01-01

    A genomic DNA-based microarray was constructed containing over 6000 randomly cloned genomic fragments of approximately 1-2 kb from six mammalian intestinal Bifidobacterium spp. including B. adolescentis, B. animalis, B. bifidum, B. catenulatum, B. longum and B. pseudolongum. This Bifidobacterium Mix

  6. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

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

    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: 1.493, year: 2014

  8. Application of microarray technology in pulmonary diseases

    Patlakas George; Tzouvelekis Argyris; Bouros Demosthenes

    2004-01-01

    Abstract Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives.

  9. OLIGONUCLEOTIDE MICROARRAY FOR THE IDENTIFICATION OF CARBAPENEMASE GENES OF MOLECULAR CLASSES A, B, AND D

    Ulyashova, M.; Khalilova, Yu; Rubtsova, M.; Edelstein, M.; Alexandrova, I; Egorov, A.

    2010-01-01

    This work is a report on the development of a method of hybridization analysis on DNA microarrays for the simultaneous identification and typing of carbapenemase-encoding genes. These enzymes are produced by the microorganisms that are responsible for causing infectious diseases. The method involves several steps, including DNA extraction from clinical samples and amplification of carbapenemase genes by multiplex PCR with simultaneous labelling by biotin. Following that, hybridization of the ...

  10. Detection of integrase in retroviral particles by a specific DNA sensor with a potential for adaptation to point-of-care diagnosis

    Wang, Jing

    2016-01-01

    The acquired immunodeficiency syndrome (AIDS) caused by HIV is one of the greatest threats to human health. Currently, HIV-infected individuals are treated by FDA approved highly active antiretroviral therapy that targets three steps of the HIV life cycle. HAART includes a protease inhibitor...... might be controlled by rational drug development and combination therapy. Early detection of HIV-positive individuals has a direct impact on therapeutic efficacy and can therefore reduce the risk of transmission. With regard to the disadvantage of the current routine HIV screening, it is therefore...... important to develop a sensitive and specific assay for the early detection, which can be adapted to point-of-care diagnosis. In the current study we describe a new specific DNA sensor system that allows the specific and sensitive detection of retrovirus IN activity. The system relies on one-end integration...

  11. Relationship between gene co-expression and probe localization on microarray slides

    Qian Jiang

    2003-12-01

    Full Text Available Abstract Background Microarray technology allows simultaneous measurement of thousands of genes in a single experiment. This is a potentially useful tool for evaluating co-expression of genes and extraction of useful functional and chromosomal structural information about genes. Results In this work we studied the association between the co-expression of genes, their location on the chromosome and their location on the microarray slides by analyzing a number of eukaryotic expression datasets, derived from the S. cerevisiae, C. elegans, and D. melanogaster. We find that in several different yeast microarray experiments the distribution of the number of gene pairs with correlated expression profiles as a function of chromosomal spacing is peaked at short separations and has two superimposed periodicities. The longer periodicity has a spacing of 22 genes (~42 Kb, and the shorter periodicity is 2 genes (~4 Kb. Conclusion The relative positioning of DNA probes on microarray slides and source plates introduces subtle but significant correlations between pairs of genes. Careful consideration of this spatial artifact is important for analysis of microarray expression data. It is particularly relevant to recent microarray analyses that suggest that co-expressed genes cluster along chromosomes or are spaced by multiples of a fixed number of genes along the chromosome.

  12. Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)

    Larkin, Patrick; Villeneuve, Daniel L.; Knoebl, Iris; Miracle, Ann L.; Carter, Barbara J.; Liu, Li; Denslow, Nancy D.; Ankley, Gerald T.

    2007-07-01

    Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol’s expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.

  13. Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

    Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

  14. Carbohydrate Microarrays in Plant Science

    Fangel, Jonatan Ulrik; Pedersen, H.L.; Vidal-Melgosa, S.;

    2012-01-01

    Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important...... industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high...... plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities....

  15. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    John S. Furey

    2005-04-01

    Full Text Available In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA and Hierarchical Cluster Analysis (HCA were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  16. Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

    Nelson Colleen C

    2005-09-01

    Full Text Available Abstract Background We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. Results We have analyzed and evaluated the utility of a microarray containing 16,006 (16K salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC library containing Atlantic salmon genomic DNA. Conclusion We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.

  17. Application of Microarray Technology and Softcomputing in Cancer Biology : A Review

    P.K.Vaishali & Dr. A.vinayababu

    2011-10-01

    Full Text Available DNA microarray technology has emerged as a boon to the scientific community in understanding thegrowth and development of life as well as in widening their knowledge in exploring the geneticcauses of anomalies occurring in the working of the human body. microarray technology makesbiologists be capable of monitoring expression of thousands of genes in a single experiment on asmall chip. Extracting useful knowledge and info from these microarray has attracted the attention ofmany biologists and computer scientists. Knowledge engineering has revolutionalized the way inwhich the medical data is being looked at. Soft computing is a branch of computer science capable ofanalyzing complex medical data. Advances in the area of microarray –based expression analysishave led to the promise of cancer diagnosis using new molecular based approaches. Many studiesand methodologies have come up which analyszes the gene espression data by using thetechniques in data mining such as feature selection, classification, clustering etc. emboiding the softcomputing methods for more accuracy. This review is an attempt to look at the recent advances incancer research with DNA microarray technology , data mining and soft computing techniques.

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

    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.

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

    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 103A. 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

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

    Lee, Dae-Young; Lauder, Heather; Cruwys, Heather; Falletta, Patricia; Beaudette, Lee A

    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(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. PMID:18423816