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Sample records for based rna extraction

  1. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

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    Lindblad Peter

    2009-08-01

    Full Text Available Abstract Background The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction. Results With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution. Conclusion It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

  2. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

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    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  3. Evaluation of DNA and RNA extraction methods.

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    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R

    2010-06-01

    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  4. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

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    Zhang Haiyu

    2007-12-01

    Full Text Available Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD, testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4 was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old" samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of

  5. Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

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    Coustham Vincent

    2011-03-01

    Full Text Available Abstract Background Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR. Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL. Results We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96 adapted from a well established phenol:chloroform-LiCl method (P:C-L that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays. Conclusion The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

  6. Extraction of Bacterial RNA from Soil: Challenges and Solutions

    OpenAIRE

    Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

    2012-01-01

    Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing techn...

  7. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

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    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  8. Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid.

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    Sun, Ning; Deng, Congliang; Zhao, Xiaoli; Zhou, Qi; Ge, Guanglu; Liu, Yi; Yan, Wenlong; Xia, Qiang

    2014-02-01

    In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection.

  9. Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore,the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied.It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

  10. Comparison of DNA and RNA extraction methods for mummified tissues.

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    Konomi, Nami; Lebwohl, Eve; Zhang, David

    2002-12-01

    Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.

  11. COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

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    ANNY CAROLYNE DA LUZ

    2016-02-01

    Full Text Available ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA, TRIzol® Reagent (Invitrogen and TRIzol® Reagent (Invitrogen/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR, and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

  12. Optimization of RNA Extraction from Rat Pancreatic Tissue

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    Sanaz Dastgheib

    2014-05-01

    Full Text Available Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1 homogenizing, 2 effective denaturation of proteins from RNA, 3 inactivation of ribonuclease, and 4 removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols. Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results. Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR. Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue

  13. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

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    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. PMID:25625965

  14. An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction.

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    Kang, Seungha; Denman, Stuart E; Morrison, Mark; Yu, Zhongtang; McSweeney, Chris S

    2009-05-01

    An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.

  15. Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks

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    Ž. Radulović

    2010-08-01

    Full Text Available A reverse transcriptase - polymerase chain reaction based assay for Borrelia species detection in ticks was developed. The method was based on amplification of 552 nucleotide bases long sequence of 16S rRNA, targeted by Borrelia specific primers. In the present study, total RNA extracted from Ixodes ricinus ticks was used as template. The results showed higher sensitivity for Borrelia detection as compared to standard dark-field microscopy. Method specificity was confirmed by cloning and sequencing of obtained 552 base pairs long amplicons. Phylogenetic analysis of obtained sequences showed that they belong to B. lusitaniae and B. afzelii genospecies. RT-PCR based method presented in this paper could be very useful as a screening test for detecting pathogen presence, especially when in investigations is required extraction of total RNA from ticks.

  16. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

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    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  17. Ultrasensitive detection of mRNA extracted from cancerous cells achieved by DNA rotaxane-based cross-rolling circle amplification.

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    Bi, Sai; Cui, Yangyang; Li, Li

    2013-01-01

    An ultrasensitive and highly selective method for polymerase chain reaction-free (PCR-free) messenger RNA (mRNA) expression profiling is developed through a novel cross-rolling circle amplification (C-RCA) process based on DNA-rotaxane nanostructures. Two species of DNA pseudorotaxane (DPR) superstructures (DPR-I and DPR-II) are assembled by threading a linear DNA rod through a double-stranded DNA (dsDNA) ring containing two single-stranded gaps. In this assay, cDNA that is specific for β-actin (ACTB) mRNA is taken as a model analyte. Upon the introduction of the target cDNA, the cDNA and the biotin-modified primer are hybridized to the single-stranded regions of the DNA rod and the gap-ring, respectively. As a result, the DPR-I dethreads into free DNA macrocycle and a dumbbell-shaped DNA nanostructure. In the presence of DNA polymerase/dNTPs, two release-DNA on the DPR-I are replaced by polymerase with strand-displacement activity, which can act as the input of the DPR-II to trigger the dethreading of DPR-II and the RCA reaction, releasing another two specified release-DNA strands those in turn serve as the "mimic cDNA" for DPR-I. The C-RCA reaction then proceeds autonomously. To overcome the high background induced by hemin itself, the biotinylated rolling circle products are captured by streptavidin-coated MNPs, achieving a detection limit as low as 0.1 zmol cDNA. The assay also exhibits an excellent selectivity due to its unique DNA nanostructure fabricated through base pairing hybridization. The ACTB mRNA expression in mammary cancer cells (MCF-7) is successfully detected.

  18. Comparison of RNA extraction methods in Thai aromatic coconut water

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    Nopporn Jaroonchon

    2015-10-01

    Full Text Available Many researches have reported that nucleic acid in coconut water is in free form and at very low yields which makes it difficult to process in molecular studies. Our research attempted to compare two extraction methods to obtain a higher yield of total RNA in aromatic coconut water and monitor its change at various fruit stages. The first method used ethanol and sodium acetate as reagents; the second method used lithium chloride. We found that extraction using only lithium chloride gave a higher total RNA yield than the method using ethanol to precipitate nucleic acid. In addition, the total RNA from both methods could be used in amplification of betaine aldehyde dehydrogenase2 (Badh2 genes, which is involved in coconut aroma biosynthesis, and could be used to perform further study as we expected. From the molecular study, the nucleic acid found in coconut water increased with fruit age.

  19. Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

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    Jailson F. B. Querido

    2013-01-01

    Full Text Available Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV, a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae, one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

  20. Extraction of total RNA in the developing chicken forebrain

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    Sayed Rasoul Zaker

    2014-01-01

    Full Text Available Background: Gene expression of Gama-Aminobutyric acid (GABA A receptor subunits may change during development. Procedures in molecular biology are required to understand the gene expression profile GABA A R in chicken. The outcome of the results depends on good-quality high-molecular-weight RNA. Several procedures can be used to isolate RNA from the brain of chicken; however, most of them are time-consuming and require disruption of cells or freeze and thaw in the presence of RNase inhibitors. The aim of this experiment was isolation of RNA from chicken embryonic brain tissues using appropriate RNA extraction kit. Materials and Methods: Fertilized eggs from Ross breed (Gallus gallus were incubated at 38°C and 60% relative humidity in a forced-draft incubator and were turned every 3 h. After 3, 7, 14 and 20 days of incubation, eggs were cooled on ice to induce deep anesthesia. Then whole brains were dissected out. As brains could not be excised in a reproducible way from earlier embryos (embryonic days 4 and 6, whole heads were collected. Chicken embryos between day 7 to 20 and 1 day after birth were decapitated, and their brains removed. Samples were immediately inserted into lysis buffer and stored at −70°C. Total RNA was isolated and a contaminating genomic deoxyribonucleic acid (DNA was digested. RNA quality was checked using gel electrophoresis. Results: We obtained 52 mg/ml to 745 mg/ml with A260/280 1.7-2.2. Only high-quality RNA, with no signs of degradation, was used for further experiments. Conclusion: In conclusion, protocol was found to be suitable for the isolation of total RNA from embryonic chicken cells.

  1. Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

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    Lee, Soo Hyeon; Kang, Yoon Young; Jang, Hyo-Eun; Mok, Hyejung

    2016-09-01

    Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications. PMID:26514375

  2. Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells

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    Gonzalez-Escalona Narjol

    2010-07-01

    Full Text Available Abstract Background The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR. Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix. Findings We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR. Conclusions We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.

  3. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    International Nuclear Information System (INIS)

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein β-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system

  4. High quality RNA extraction from Maqui berry for its application in next-generation sequencing.

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    Sánchez, Carolina; Villacreses, Javier; Blanc, Noelle; Espinoza, Loreto; Martinez, Camila; Pastor, Gabriela; Manque, Patricio; Undurraga, Soledad F; Polanco, Victor

    2016-01-01

    Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.

  5. High quality RNA extraction from Maqui berry for its application in next-generation sequencing.

    Science.gov (United States)

    Sánchez, Carolina; Villacreses, Javier; Blanc, Noelle; Espinoza, Loreto; Martinez, Camila; Pastor, Gabriela; Manque, Patricio; Undurraga, Soledad F; Polanco, Victor

    2016-01-01

    Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins. PMID:27536526

  6. ncRNA-class Web Tool: Non-coding RNA feature extraction and pre-miRNA classification web tool

    KAUST Repository

    Kleftogiannis, Dimitrios A.

    2012-01-01

    Until recently, it was commonly accepted that most genetic information is transacted by proteins. Recent evidence suggests that the majority of the genomes of mammals and other complex organisms are in fact transcribed into non-coding RNAs (ncRNAs), many of which are alternatively spliced and/or processed into smaller products. Non coding RNA genes analysis requires the calculation of several sequential, thermodynamical and structural features. Many independent tools have already been developed for the efficient calculation of such features but to the best of our knowledge there does not exist any integrative approach for this task. The most significant amount of existing work is related to the miRNA class of non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that play a significant role in gene regulation and their prediction is a challenging bioinformatics problem. Non-coding RNA feature extraction and pre-miRNA classification Web Tool (ncRNA-class Web Tool) is a publicly available web tool ( http://150.140.142.24:82/Default.aspx ) which provides a user friendly and efficient environment for the effective calculation of a set of 58 sequential, thermodynamical and structural features of non-coding RNAs, plus a tool for the accurate prediction of miRNAs. © 2012 IFIP International Federation for Information Processing.

  7. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Melissa A McAlexander

    2013-05-01

    Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

  8. 高温弱碱稀盐法从啤酒废酵母中提取RNA%Extracting RNA from spent brewer's yeast by the new technology rare salt of high temperature of weak base

    Institute of Scientific and Technical Information of China (English)

    何娟; 詹自力; 夏萍; 曹颖; 卢奎

    2006-01-01

    采用盐法与碱法相结合破壁的方法,即高温弱碱稀盐法从啤酒废酵母中提取核糖核酸(RNA).正交试验确定的最佳工艺条件为:抽提温度100 ℃,w(NaCl)=6%,抽提时间90 min,pH=8.在此条件下,RNA收率可达8.01%,较传统工艺收率有显著提高.

  9. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  10. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    International Nuclear Information System (INIS)

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ∼90% for deoxyribonucleic acid (DNA) and ∼54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components

  11. Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

    OpenAIRE

    Schultz, M C; Choe, S Y; Reeder, R H

    1991-01-01

    A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast.

  12. A modified protocol for RNA extraction from different peach tissues suitable for gene isolation and real-time PCR analysis.

    Science.gov (United States)

    Tong, Zhaoguo; Qu, Shenchun; Zhang, Jiyu; Wang, Fei; Tao, Jianmin; Gao, Zhihong; Zhang, Zhen

    2012-03-01

    RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods, we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as from other tissues including flowers, leaves, stems, and roots. With our modified method, 28-650 μg of total RNA was routinely obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR.

  13. qPCR based mRNA quality score show intact mRNA after heat stabilization

    Directory of Open Access Journals (Sweden)

    Oskar Karlsson

    2016-03-01

    Full Text Available Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

  14. RNA preservation agents and nucleic acid extraction method bias perceived bacterial community composition.

    Directory of Open Access Journals (Sweden)

    Ann McCarthy

    Full Text Available Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA

  15. Nonenzymatic microorganism identification based on ribosomal RNA

    Science.gov (United States)

    Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

    1999-11-01

    Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

  16. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    OpenAIRE

    Diaz, Patricia I.; Abusleme, Loreto; Hong, Bo-Young; Amanda K. Dupuy; Linda D Strausbaugh

    2014-01-01

    Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction proced...

  17. Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    Hélène Pelczar

    Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

  18. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

    NARCIS (Netherlands)

    Duarte, G.F.; Rosado, A.S.; Keijzer-Wolters, A.C.; Elsas, van J.D.

    1998-01-01

    A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils

  19. Information based universal feature extraction

    Science.gov (United States)

    Amiri, Mohammad; Brause, Rüdiger

    2015-02-01

    In many real world image based pattern recognition tasks, the extraction and usage of task-relevant features are the most crucial part of the diagnosis. In the standard approach, they mostly remain task-specific, although humans who perform such a task always use the same image features, trained in early childhood. It seems that universal feature sets exist, but they are not yet systematically found. In our contribution, we tried to find those universal image feature sets that are valuable for most image related tasks. In our approach, we trained a neural network by natural and non-natural images of objects and background, using a Shannon information-based algorithm and learning constraints. The goal was to extract those features that give the most valuable information for classification of visual objects hand-written digits. This will give a good start and performance increase for all other image learning tasks, implementing a transfer learning approach. As result, in our case we found that we could indeed extract features which are valid in all three kinds of tasks.

  20. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

    Science.gov (United States)

    Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael

    2016-10-01

    The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS). PMID:27387642

  1. Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

    OpenAIRE

    Gea Guerriero; Lauralie Mangeot-Peter; Jean-Francois Hausman; Sylvain Legay

    2016-01-01

    In plants there is no universal protocol for RNA extraction, since optimizations are required depending on the species, tissues and developmental stages. Some plants/tissues are rich in secondary metabolites or synthesize thick cell walls, which hinder an efficient RNA extraction. One such example is bast fibres, long extraxylary cells characterized by a thick cellulosic cell wall. Given the economic importance of bast fibres, which are used in the textile sector, as well as in biocomposites ...

  2. Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

    OpenAIRE

    Gisele Rodrigues Gouveia; Suzete Cleusa Ferreira; Jerenice Esdras Ferreira; Sheila Aparecida Coelho Siqueira; Juliana Pereira

    2014-01-01

    The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the Recov...

  3. Simultaneous Extraction of DNA and RNA from Animal Cells by a Modified Laemmli Buffer

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Nasrin Shojaie, Mahmood S. Ghaffari & Zahra Safari ### Abstract Simultaneous investigation of DNA and RNA is often a necessity in genetic manipulation and biological researches. Besides, most of the traditional procedures devised for RNA isolation have some difficulties associated with RNase activity. Therefore, this protocol presents a safer process to extract high purity RNA in shorter time. ### Introduction The protocol describes a simple and less time-consumi...

  4. On-Orbit DNA, RNA, and Protein Extraction Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Genova Engineering proposes to develop and demonstrate a toolset of discrete devices and extraction kits which will leverage existing on-orbit facilities and will...

  5. 玉米矮花叶病毒CP基因dsRNA的原核表达与分离%Prokaryotic Expression and Extraction of dsRNA Based on the CP Gene of Maize Dwarf Mosaic Virus

    Institute of Scientific and Technical Information of China (English)

    甘德芳; 张姣; 赵阳; 朱苏文; 程备久

    2011-01-01

    根据玉米矮花叶病毒CP基因序列设计特异性引物,RT-PCR扩增玉米矮花叶病毒CP基因特异性干涉片段,将干涉片段及pUCCRNAi载体分别用BamH I及Sal I双酶切,然后将干涉片段分别正反向插入pUC- CRNAi载体中,构建CP基因反向重复克隆载体pUCCRNAi+2 F.再利用Pst I-Sal I位点插入到L4440质粒中构建原核表达载体LMCP.利用IPTG进行诱导表达并对诱导表达条件进行优化.结果表明,经过IPTG诱导,LMCP在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase I和RNase A消化处理,证实为dsRNA.同时IPTG浓度为0.4~0.6 mmol/L,诱导表达4 h,dsRNA的表达量最高.另外,溶解于ddH2O中的dsRNA稳定性要高于溶解在NaCl中的,且随着放置时间的延长,dsRNA将出现明显的降解.%MDMV CP gene fragments were amplified by RT-PCR from extracted MDMV mRNA. To prepare a hairpin RNA, MDMV CP gene fragments and the pUCCRNAi cloning vector were digested by BamH I-Sal I respectively, First,the BamH I-Sal I fragment from MDMV RNA was cloned in the positive orientation into pUCCRNAi to generate pUCCRNAi + F. And then, the other BamH I-Sal I fragment was cloned in the reverse orientation into Bgl II-Xho I digested pUCCRNAi + F to generate an inverted repeat sequence of pUCCRNAi + 2 F ( sense orientation fragment and antisense orientation fragment were separated by an intron). Thirdly, L4440 and pUCCRNAi + 2 F plasmids were digested with Pst I-Sal I and subsequently joined to generate LMCP. And the recombinant plasmid was induced by IPTG. The results showed that the expression product was the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of 0.4 ~ 0.6 mmol/L and induction time of 4 h was the most optimal expression condition. The stability of the dsRNA in ddH20 is higher than that of in NaCl, and the dsRNA appeares to he dissolved with the time extending.

  6. RNA extraction from plant tissues: the use of calcium to precipitate contaminating pectic sugars.

    Science.gov (United States)

    Dal Cin, Valeriano; Danesin, Marcello; Rizzini, Fabio Massimo; Ramina, Angelo

    2005-10-01

    Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca2+, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.

  7. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    Science.gov (United States)

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. PMID:27237373

  8. Web-Based Information Extraction Technology

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Information extraction techniques on the Web are the current research hotspot. Now many information extraction techniques based on different principles have appeared and have different capabilities. We classify the existing information extraction techniques by the principle of information extraction and analyze the methods and principles of semantic information adding, schema defining,rule expression, semantic items locating and object locating in the approaches. Based on the above survey and analysis,several open problems are discussed.

  9. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Loreto Abusleme

    2014-04-01

    Full Text Available Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the

  10. A microfluidic approach for high efficiency extraction of low molecular weight RNA.

    Science.gov (United States)

    Vulto, Paul; Dame, Gregory; Maier, Urban; Makohliso, Solomzi; Podszun, Susann; Zahn, Peter; Urban, Gerald A

    2010-03-01

    The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a

  11. iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

    Directory of Open Access Journals (Sweden)

    Jittrawan Thaiprasit

    2016-03-01

    Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

  12. RNA-Based Vaccines in Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Megan A. McNamara

    2015-01-01

    Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

  13. RNA-Based Vaccines in Cancer Immunotherapy.

    Science.gov (United States)

    McNamara, Megan A; Nair, Smita K; Holl, Eda K

    2015-01-01

    RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

  14. Evaluation of Six Methods for Extraction of Total RNA from Loquat

    Directory of Open Access Journals (Sweden)

    Zhang LING

    2013-05-01

    Full Text Available   Trizol extraction for polysaccharide-rich plant tissue was unsuitable for isolating total RNA from loquat fruit. CTAB-LiCl extraction was improved with pretreatment using washing buffer with 80% ethanol and 70% acetone. The RNA isolated by this protocol from different loquat fruit tissue at various development periods, was high in purity (A260/A280 ratio ranged from 1.81 to 1.99 and A260/A230 ratio was over 2.0, and high in yield.

  15. Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

    DEFF Research Database (Denmark)

    Paulin, Mélanie Marie; Nicolaisen, Mette Haubjerg; Jacobsen, Carsten Suhr;

    2013-01-01

    -time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca in combination with high p......Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved...... extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real...

  16. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    Science.gov (United States)

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-06-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

  17. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    DEFF Research Database (Denmark)

    Lever, Mark; Torti, Andrea; Eickenbusch, Philip;

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate......'s oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular...... tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world...

  18. Theoretical analysis of noncanonical base pairing interactions in RNA molecules

    Indian Academy of Sciences (India)

    Dhananjay Bhattacharyya; Siv Chand Koripella; Abhijit Mitra; Vijay Babu Rajendran; Bhabdyuti Sinha

    2007-08-01

    Noncanonical base pairs in RNA have strong structural and functional implications but are currently not considered for secondary structure predictions. We present results of comparative ab initio studies of stabilities and interaction energies for the three standard and 24 selected unusual RNA base pairs reported in the literature. Hydrogen added models of isolated base pairs, with heavy atoms frozen in their ‘away from equilibrium’ geometries, built from coordinates extracted from NDB, were geometry optimized using HF/6-31G** basis set, both before and after unfreezing the heavy atoms. Interaction energies, including BSSE and deformation energy corrections, were calculated, compared with respective single point MP2 energies, and correlated with occurrence frequencies and with types and geometries of hydrogen bonding interactions. Systems having two or more N-H…O/N hydrogen bonds had reasonable interaction energies which correlated well with respective occurrence frequencies and highlighted the possibility of some of them playing important roles in improved secondary structure prediction methods. Several of the remaining base pairs with one N-H…O/N and/or one C-H…O/N interactions respectively, had poor interaction energies and negligible occurrences. High geometry variations on optimization of some of these were suggestive of their conformational switch like characteristics.

  19. Total RNA extraction from strawberry tree (Arbutus unedo and several other woody-plants

    Directory of Open Access Journals (Sweden)

    Zamboni A

    2008-08-01

    Full Text Available Studies of plant gene expression today need pure preparations of high-yielding undegraded RNA. This is not easily accomplished when working with plants and tissues like strawberry tree (Arbutus unedo leaves that accumulate large amounts of polysaccharides and polyphenolic compounds, which co-purify with RNA. An improved leaf-tissue protocol developed for gene expression studies on Arbutus unedo yields for the first time a purity of RNA extract that makes possible cDNA synthesis and qPCR analysis in this plant species. When tested on material considered recalcitrant (leaves, roots, fruit flesh, fruit peel and styles from Pyrus communis, Prunus avium, Prunus persica and Cydonia oblonga, the method was able to extract RNA with good yield and high purity. This scalable, phenol-free, fast and easy-to-use RNA extraction protocol is effective on Arbutus unedo leaves as well as on awide range of different species and tissues, thus resulting particularly useful for gene expression analysis in non-model species for molecular biology.

  20. Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

    Directory of Open Access Journals (Sweden)

    Gisele Rodrigues Gouveia

    2014-01-01

    Full Text Available The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE specimens of diffuse large B-cell lymphoma (DLBCL. We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion. However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens.

  1. Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

    Directory of Open Access Journals (Sweden)

    Gea Guerriero

    2016-07-01

    Full Text Available In plants there is no universal protocol for RNA extraction, since optimizations are required depending on the species, tissues and developmental stages. Some plants/tissues are rich in secondary metabolites or synthesize thick cell walls, which hinder an efficient RNA extraction. One such example is bast fibres, long extraxylary cells characterized by a thick cellulosic cell wall. Given the economic importance of bast fibres, which are used in the textile sector, as well as in biocomposites as green substitutes of glass fibres, it is desirable to better understand their development from a molecular point of view. This knowledge favours the development of biotechnological strategies aimed at improving specific properties of bast fibres. To be able to perform high-throughput analyses, such as, for instance, transcriptomics of bast fibres, RNA extraction is a crucial and limiting step. We here detail a protocol enabling the rapid extraction of high quality RNA from the bast fibres of textile hemp, Cannabis sativa L., a multi-purpose fibre crop standing in the spotlight of research.

  2. Extraction of mRNA from coagulated horse blood and analysis of inflammation-related cytokine responses to coagulation

    DEFF Research Database (Denmark)

    Bovbjerg, Kirsten Katrine Lindegaard; Heegaard, Peter M. H.; Skovgaard, Kerstin

    2010-01-01

    available. Here, a protocol for RNA extraction from highly clotted blood was optimized and the regulation of a number of cytokine genes compared to stabilized blood was studied. Whole blood samples from 10 clinically healthy horses were incubated for 24 hours at 37°C and RNA was extracted from...

  3. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    Science.gov (United States)

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  4. Chinese Term Extraction Based on PAT Tree

    Institute of Scientific and Technical Information of China (English)

    ZHANG Feng; FAN Xiao-zhong; XU Yun

    2006-01-01

    A new method of automatic Chinese term extraction is proposed based on Patricia (PAT) tree. Mutual information is calculated based on prefix searching in PAT tree of domain corpus to estimate the internal associative strength between Chinese characters in a string. It can improve the speed of term candidate extraction largely compared with methods based on domain corpus directly. Common collocation suffix, prefix bank are constructed and term part of speech (POS) composing rules are summarized to improve the precision of term extraction. Experiment results show that the F-measure is 74.97 %.

  5. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    Science.gov (United States)

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

  6. Comparison of several methods of total RNA extraction for Candida tropicalis%几种提取热带假丝酵母总RNA方法比较

    Institute of Scientific and Technical Information of China (English)

    龙燕; 杨升

    2012-01-01

    Three methods including Trizol method, hot-phenol method, and Yeast RNA kit method were used for the total RNA extraction from Candida tropicalis. Based on total RNA concentration and purity, these methods were compared for their effects on total RNA extraction from Candida tropicalis. The results show the Yeast RNA kit method is the most effective and convenient method for total RNA extraction from Candida tropicalis.%选择Trizol法、热酚法、酵母RNA提取试剂盒法3种方法提取热带假丝酵母总RNA,通过对总RNA进行质量浓度和纯度分析,比较不同方法对热带假丝酵母总RNA提取的影响.结果表明:使用酵母RNA提取试剂盒法提取热带假丝酵母总RNA是较为有效且简便的方法.

  7. Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

    OpenAIRE

    Ruskin, B; Pikielny, C W; Rosbash, M; Green, M R

    1986-01-01

    Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the...

  8. miRNA-Based Therapeutic Strategies

    OpenAIRE

    Ishida, Masaharu; Selaru, Florin M.

    2012-01-01

    Micro-RNAs (miRNAs) are short non-coding RNA species, thought to act primarily through downregulation of target mRNA species with subsequent decrease in encoded proteins. Recent studies revealed that miRNAs play pivotal roles in physiology and disease, and therapeutic targeting has started being investigated. Generally, the up-regulation of miRNAs is achieved through administration of synthetic miRNAs or administration of miRNA expressing vectors. The down-regulation of miRNAs is achieved thr...

  9. Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep.

    Science.gov (United States)

    Mura, Maria Consuelo; Daga, Cinzia; Bodano, Sara; Paludo, Marta; Luridiana, Sebastiano; Pazzola, Michele; Dettori, Maria Luisa; Vacca, Giuseppe Massimo; Carcangiu, Vincenzo

    2013-03-01

    The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.

  10. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

    Directory of Open Access Journals (Sweden)

    Adam Kotorashvili

    Full Text Available BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of

  11. A cell-free extract from yeast cells for studying mRNA turnover.

    OpenAIRE

    Vreken, P.; Buddelmeijer, N.; Raué, H A

    1992-01-01

    We have isolated a cell-free extract from yeast cells that reproduces the differences observed in vivo in the rate of turnover of individual yeast mRNAs. Detailed analysis of the degradation of yeast phosphoglycerate kinase (PGK) mRNA in this system demonstrated that both natural and synthetically prepared PGK transcripts are degraded by the same pathway previously established by us in vivo, consisting of endonucleolytic cleavage at a number of 5'-GGUG-3' sequence motifs within a short target...

  12. Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

    Directory of Open Access Journals (Sweden)

    Schwochow Doreen

    2012-06-01

    Full Text Available Abstract Background Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores. Results Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg with varying integrity (RIN 4.6 - 7.7, the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing. Conclusion By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.

  13. Discovering functional modules by topic modeling RNA-Seq based toxicogenomic data.

    Science.gov (United States)

    Yu, Ke; Gong, Binsheng; Lee, Mikyung; Liu, Zhichao; Xu, Joshua; Perkins, Roger; Tong, Weida

    2014-09-15

    Toxicogenomics (TGx) endeavors to elucidate the underlying molecular mechanisms through exploring gene expression profiles in response to toxic substances. Recently, RNA-Seq is increasingly regarded as a more powerful alternative to microarrays in TGx studies. However, realizing RNA-Seq's full potential requires novel approaches to extracting information from the complex TGx data. Considering read counts as the number of times a word occurs in a document, gene expression profiles from RNA-Seq are analogous to a word by document matrix used in text mining. Topic modeling aiming at to discover the latent structures in text corpora would be helpful to explore RNA-Seq based TGx data. In this study, topic modeling was applied on a typical RNA-Seq based TGx data set to discover hidden functional modules. The RNA-Seq based gene expression profiles were transformed into "documents", on which latent Dirichlet allocation (LDA) was used to build a topic model. We found samples treated by the compounds with the same modes of actions (MoAs) could be clustered based on topic similarities. The topic most relevant to each cluster was identified as a "marker" topic, which was interpreted by gene enrichment analysis with MoAs then confirmed by compound and pathways associations mined from literature. To further validate the "marker" topics, we tested topic transferability from RNA-Seq to microarrays. The RNA-Seq based gene expression profile of a topic specifically associated with peroxisome proliferator-activated receptors (PPAR) signaling pathway was used to query samples with similar expression profiles in two different microarray data sets, yielding accuracy of about 85%. This proof-of-concept study demonstrates the applicability of topic modeling to discover functional modules in RNA-Seq data and suggests a valuable computational tool for leveraging information within TGx data in RNA-Seq era. PMID:25083553

  14. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    Science.gov (United States)

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC). PMID:27045556

  15. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    Science.gov (United States)

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-01-01

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

  16. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    Science.gov (United States)

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-08-21

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

  17. Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis.

    Directory of Open Access Journals (Sweden)

    Karl Kashofer

    Full Text Available Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek, and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre

  18. Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

    Directory of Open Access Journals (Sweden)

    Vincent R. Hill

    2015-05-01

    Full Text Available Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

  19. Magnetic Resonance Spectroscopy of siRNA-Based Cancer Therapy

    Science.gov (United States)

    Penet, Marie-France; Chen, Zhihang; Mori, Noriko; Krishnamachary, Balaji; Bhujwalla, Zaver M.

    2016-01-01

    Small interfering RNA (siRNA) is routinely used as a biological tool to silence specific genes, and is under active investigation in cancer treatment strategies. Noninvasive magnetic resonance spectroscopy (MRS) provides the ability to assess the functional effects of siRNA-mediated gene silencing in cultured cancer cells, and following nanoparticle-based delivery in tumors in vivo. Here we describe the use of siRNA to downregulate choline kinase, a critical enzyme in choline phospholipid metabolism of cancer cells and tumors, and the use of 1H MRS of cells and 1H magnetic resonance spectroscopic imaging (MRSI) of tumors to assess the efficacy of the downregulation. PMID:26530913

  20. In situ, real-time catabolic gene expression: Extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater

    International Nuclear Information System (INIS)

    The authors developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen to dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To the authors' knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches

  1. 蟠桃果实总RNA提取方法的研究%RNA Extraction Method from Mature Flat Peach Fruits

    Institute of Scientific and Technical Information of China (English)

    史元敏; 何天明; 袁海英

    2012-01-01

    [Objective]The target of current research was to select and obtain a proper RNA extraction method from flat peach fruit. [ Method ] RNAplant plus, RNAisoTM, improved SDS method and improved CTAB method were used to isolate total RNA from flat peach fruits harvested at 100 days after full blossom. [Result]RNA extracted by two extraction kits had poor quality with contamination of the genome DNA; RNA extracted by improved SDS method contained large amounts of polysaccharide, while RNA extracted by the improved CTAB method had good integrity and the RNA bands were clear without degradation. RT - PCR and Northern blot test indicated that the extracted RNA could be used for die following molecular biology procedures. [ Conclusion ] The establishment of optimum RNA extraction mediod from flat peach fruits -improved CTAB method the base for the follow - up molecular biology study of fruit development.%[目的]筛选获得提取蟠桃果肉中总RNA的适宜方法.[方法]以盛花后100 d的蟠桃为材料,采用Transplant plus植物总RNA提取试剂提取法、RNAisoTM试剂盒法、改良SDS法、改良CTAB法等四种方法提取总RNA.[结果]两种试剂盒法提取的总RNA质量差,有严重的基因组污染;改良SDS法提取物中含有大量的多糖;采用改良CTAB法所得RNA完整性好,条带清晰无降解,无基因组污染.经过RT - PCR和Northern blot检测后,表明该RNA能够满足后续分子生物学操作的要求.[结论]蟠桃果实总RNA适宜提取方法-改良CTAB法的确定为后续果实发育分子生物学的研究奠定了基础.

  2. RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device.

    Science.gov (United States)

    Ma, Sai; Bryson, Bryan D; Sun, Chen; Fortune, Sarah M; Lu, Chang

    2016-05-17

    Studies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000-8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10-20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays. PMID:27081872

  3. Comparative evaluation of commercially available manual and automated nucleic acid extraction methods for rotavirus RNA detection in stools.

    Science.gov (United States)

    Esona, Mathew D; McDonald, Sharla; Kamili, Shifaq; Kerin, Tara; Gautam, Rashi; Bowen, Michael D

    2013-12-01

    Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS easyMAG instruments, the NucliSENS miniMAG semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols. PMID:24036075

  4. Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

    Science.gov (United States)

    Lee, Jonghwan; Kim, Soonhag

    2016-01-01

    The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h. PMID:26530921

  5. Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

    Science.gov (United States)

    Lee, Jonghwan; Kim, Soonhag

    2016-01-01

    The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

  6. Facial Feature Extraction Based on Wavelet Transform

    Science.gov (United States)

    Hung, Nguyen Viet

    Facial feature extraction is one of the most important processes in face recognition, expression recognition and face detection. The aims of facial feature extraction are eye location, shape of eyes, eye brow, mouth, head boundary, face boundary, chin and so on. The purpose of this paper is to develop an automatic facial feature extraction system, which is able to identify the eye location, the detailed shape of eyes and mouth, chin and inner boundary from facial images. This system not only extracts the location information of the eyes, but also estimates four important points in each eye, which helps us to rebuild the eye shape. To model mouth shape, mouth extraction gives us both mouth location and two corners of mouth, top and bottom lips. From inner boundary we obtain and chin, we have face boundary. Based on wavelet features, we can reduce the noise from the input image and detect edge information. In order to extract eyes, mouth, inner boundary, we combine wavelet features and facial character to design these algorithms for finding midpoint, eye's coordinates, four important eye's points, mouth's coordinates, four important mouth's points, chin coordinate and then inner boundary. The developed system is tested on Yale Faces and Pedagogy student's faces.

  7. Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

    Science.gov (United States)

    Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

    2015-01-01

    STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. PMID:25284026

  8. Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    Science.gov (United States)

    Yang, Jian; Hotz, Tremearne; Broadnax, LaCassidy; Yarmarkovich, Mark; Elbaz-Younes, Ismail; Hirschi, Kendal D.

    2016-01-01

    Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion chromatography, and proteinase K treatment of plant extracts suggest this RNA resides within a proteinase K-sensitive complex. Plant derived MIR2911 was more bioavailable than the synthetic RNA. Furthermore, MIR2911 exhibited unusual digestive stability compared with other synthetic plant microRNAs. The characteristics of circulating MIR2911 were also unusual as it was not associated with exosomes and fractionated as a soluble complex that was insensitive to proteinase K treatment, consistent with MIR2911 being stabilized by modifications conferred by the host. These results indicate that intrinsic stability and plant-based modifications orchestrate consumer uptake of this anomalous plant based small RNA and invite revisiting plant-based microRNA therapeutic approaches. PMID:27251858

  9. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    Science.gov (United States)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  10. Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Nadhim Bayatti

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses

  11. RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris.

    Science.gov (United States)

    Weinstein, J D; Beale, S I

    1985-05-15

    Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.

  12. Combinatorics of RNA Secondary Structures with Base Triples.

    Science.gov (United States)

    Müller, Robert; Nebel, Markus E

    2015-07-01

    The structure of RNA has been the subject of intense research over the last decades due to its importance for the correct functioning of RNA molecules in biological processes. Hence, a large number of models for RNA folding and corresponding algorithms for structure prediction have been developed. However, previous models often only consider base pairs, although every base is capable of up to three edge-to-edge interactions with other bases. Recently, Höner zu Siederdissen et al. presented an extended model of RNA secondary structure, including base triples together with a folding algorithm-the first thermodynamics-based algorithm that allows the prediction of secondary structures with base triples. In this article, we investigate the search space processed by this new algorithm, that is, the combinatorics of extended RNA secondary structures with base triples. We present generalized definitions for structural motifs like hairpins, stems, bulges, or interior loops occurring in structures with base triples. Furthermore, we prove precise asymptotic results for the number of different structures (size of search space) and expectations for various parameters associated with structural motifs (typical shape of folding). Our analysis shows that the asymptotic number of secondary structures of size n increases exponentially to [Formula: see text] compared to the classic model by Stein and Waterman for which [Formula: see text] structures exist. A comparison with the classic model reveals large deviations in the expected structural appearance, too. The inclusion of base triples constitutes a significant refinement of the combinatorial model of RNA secondary structure, which, by our findings, is quantitatively characterized. Our results are of special theoretical interest, because a closer look at the numbers involved suggests that extended RNA secondary structures constitute a new combinatorial class not bijective with any other combinatorial objects studied so far.

  13. Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

    Directory of Open Access Journals (Sweden)

    Kazuya Terasawa

    2011-01-01

    Full Text Available RNA interference (RNAi is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs and DNA vector-based short hairpin RNAs (shRNAs are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

  14. Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boilign vs. conventional RNA extraction

    International Nuclear Information System (INIS)

    Full text: RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commerical lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTaPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing material materials, including diluted virus positive allontoic fluid or cell culture supernatnat, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. (author)

  15. Screening of total RNA extraction methods for RNA-sequences from different organs inZiziphus jujuba%适于转录组测序的枣不同器官总RNA提取方法筛选

    Institute of Scientific and Technical Information of China (English)

    魏琦琦; 冯延芝; 林青; 贾宝光; 张琳

    2015-01-01

    In order to obtain themethodsfor high-quality total RNA extractionfromlfower,fruit bearing branchandfruit inZiziphus jujuba, takingZ. jujuba cv.'Zhongqiusucui'asmaterials,and the RNA extraction effectswere compared using twokinds of total RNA extractionkits (Ambiogenand Autolab)andmodiifed CTABmethod based on the Autolab RNA extractionkit. Theresults showed thatOD260/OD280values of the RNA extracted by using the three RNA extractionmethods had no signiifcant differences, buta part of RNAs obtained by the two RNA extractionkitswere degraded. The RNAmass concentrationsfromlfower,fruit bearing branchandfruitwere 126, 284and 222 ng/μL by using the RNA extractionkit of Ambiogen; the RNAmass concentrationswere 307, 402and 266 ng/μL by using the RNA extractionkit of Autolab;and the RNAmass concentrationswere 401, 417and 296 ng/μL by using themodiifed CTABmethod based on the Autolab RNA extractionkit,respectively. Themodiifed CTABmethod couldgenerate high-integrity, high-concentrationand high-purity RNA that couldmeet therequest of transcriptome sequencing, comparedwith the two extractionkits.%为获得适用于转录组测序的枣花、结果枝和果实的总RNA提取方法,以中秋酥脆枣为材料,比较分析了Ambiogen和Autolab 2种总RNA提取试剂盒和基于Autolab试剂盒改良的CTAB法的总RNA的提取效果.结果表明:3种方法提取RNA的OD260/OD280差别不大,但用2种试剂盒提取的RNA有降解,其中Ambiogen试剂盒提取的花、结果枝和果实3个器官的RNA质量浓度分别为126、284和222 ng/μL;Autolab试剂盒提取的RNA质量浓度分别为307、402和266 ng/μL;基于Autolab试剂盒改良的CTAB法提取的RNA质量浓度分别为401、417和296 ng/μL.与2种试剂盒相比,基于Autolab试剂盒改良的CTAB法提取出来的RNA完整性好、纯度和浓度高,能够满足转录组测序的要求.

  16. A path-based measurement for human miRNA functional similarities using miRNA-disease associations

    Science.gov (United States)

    Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

    2016-09-01

    Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

  17. Statistical feature extraction based iris recognition system

    Indian Academy of Sciences (India)

    ATUL BANSAL; RAVINDER AGARWAL; R K SHARMA

    2016-05-01

    Iris recognition systems have been proposed by numerous researchers using different feature extraction techniques for accurate and reliable biometric authentication. In this paper, a statistical feature extraction technique based on correlation between adjacent pixels has been proposed and implemented. Hamming distance based metric has been used for matching. Performance of the proposed iris recognition system (IRS) has been measured by recording false acceptance rate (FAR) and false rejection rate (FRR) at differentthresholds in the distance metric. System performance has been evaluated by computing statistical features along two directions, namely, radial direction of circular iris region and angular direction extending from pupil tosclera. Experiments have also been conducted to study the effect of number of statistical parameters on FAR and FRR. Results obtained from the experiments based on different set of statistical features of iris images show thatthere is a significant improvement in equal error rate (EER) when number of statistical parameters for feature extraction is increased from three to six. Further, it has also been found that increasing radial/angular resolution,with normalization in place, improves EER for proposed iris recognition system

  18. EXTRACT

    DEFF Research Database (Denmark)

    Pafilis, Evangelos; Buttigieg, Pier Luigi; Ferrell, Barbra;

    2016-01-01

    therefore developed an interactive annotation tool, EXTRACT, which helps curators identify and extract standard-compliant terms for annotation of metagenomic records and other samples. Behind its web-based user interface, the system combines published methods for named entity recognition of environment...... and text-mining-assisted curation revealed that EXTRACT speeds up annotation by 15-25% and helps curators to detect terms that would otherwise have been missed.Database URL: https://extract.hcmr.gr/....

  19. Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen.

    Science.gov (United States)

    Christopher-Hennings, Jane; Dammen, Matthew; Nelson, Eric; Rowland, Raymond; Oberst, Richard

    2006-09-01

    To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen. PMID:16621036

  20. Simultaneous extraction from clinical biopsies of high-molecular-weight DNA and RNA: comparative characterization by biotinylated and 32P-labeled probes on Southern and Northern blots

    International Nuclear Information System (INIS)

    A method for efficient simultaneous extraction of high-molecular-weight DNA and RNA from solid mammalian tissues including clinical biopsies is described. It is based on the disruption and subsequent melting of deep frozen tissue in the presence of frozen phenol and nucleic acid extraction buffer; this allows for simultaneous disruption of tissue and inactivation of nucleases. The yield is about 0.7-5.8 mg of DNA and 0.5-8.1 mg of total RNA/g of tissue depending upon the tissue type; this is higher than the yield of other methods tested. Analysis of total RNA by denaturing gel electrophoresis, and of DNA and poly(A)+ RNA by Southern and Northern blot hybridization using 32P and biotinylated probes, indicated that c-Ha-ras gene and its transcripts were undegraded. Biotinylated and 32P probes had approximately the same sensitivity in detecting nucleic acids on Southern and Northern blots. This extraction procedure is simple and, when used with biotinylated probes, is rapid, inexpensive, and nonhazardous. The methodology can be modified for use with other clinical samples and cells grown in culture

  1. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    Directory of Open Access Journals (Sweden)

    Mark Alexander Lever

    2015-05-01

    Full Text Available A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world’s oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.

  2. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    Science.gov (United States)

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  3. SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

    Directory of Open Access Journals (Sweden)

    Francesca Malentacchi

    Full Text Available One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

  4. A RNA-based nanodevice recording temperature over time

    Science.gov (United States)

    Höfinger, Siegfried; Zerbetto, Francesco

    2010-04-01

    Nucleic acids provide a wealth of interesting properties that can find important applications in nanotechnology. In this article we describe a concept of how to use RNA for temperature measurements. In particular the principal components of a nanodevice are outlined that works on the basis of RNA secondary structure rearrangement. The major mode of operation is a hairpin-coil transition occurring at different temperatures for different types of short RNA oligonucleotides. Coupling these events to a detection system based on specific RNA hybridization provides the framework for a nanodevice capable of temperature records as a function of time. The analysis is carried out with the help of a statistical mechanics package that has been specifically designed to study RNA secondary structure. The procedure yields an optimized list of eight RNA sequences operational in the range from -10 to 60 °C. The data can form the basis of a new technology of potential interest to many fields of process and quality control.

  5. Inferring noncoding RNA families and classes by means of genome-scale structure-based clustering.

    Directory of Open Access Journals (Sweden)

    Sebastian Will

    2007-04-01

    Full Text Available The RFAM database defines families of ncRNAs by means of sequence similarities that are sufficient to establish homology. In some cases, such as microRNAs and box H/ACA snoRNAs, functional commonalities define classes of RNAs that are characterized by structural similarities, and typically consist of multiple RNA families. Recent advances in high-throughput transcriptomics and comparative genomics have produced very large sets of putative noncoding RNAs and regulatory RNA signals. For many of them, evidence for stabilizing selection acting on their secondary structures has been derived, and at least approximate models of their structures have been computed. The overwhelming majority of these hypothetical RNAs cannot be assigned to established families or classes. We present here a structure-based clustering approach that is capable of extracting putative RNA classes from genome-wide surveys for structured RNAs. The LocARNA (local alignment of RNA tool implements a novel variant of the Sankoff algorithm that is sufficiently fast to deal with several thousand candidate sequences. The method is also robust against false positive predictions, i.e., a contamination of the input data with unstructured or nonconserved sequences. We have successfully tested the LocARNA-based clustering approach on the sequences of the RFAM-seed alignments. Furthermore, we have applied it to a previously published set of 3,332 predicted structured elements in the Ciona intestinalis genome (Missal K, Rose D, Stadler PF (2005 Noncoding RNAs in Ciona intestinalis. Bioinformatics 21 (Supplement 2: i77-i78. In addition to recovering, e.g., tRNAs as a structure-based class, the method identifies several RNA families, including microRNA and snoRNA candidates, and suggests several novel classes of ncRNAs for which to date no representative has been experimentally characterized.

  6. RNA-Based Assessment of Diversity and Composition of Active Archaeal Communities in the German Bight

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer

    2012-01-01

    Full Text Available Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota and the Marine Group I (Thaumarchaeota were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

  7. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    Science.gov (United States)

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  8. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    Science.gov (United States)

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  9. Building Extraction from LIDAR Based Semantic Analysis

    Institute of Scientific and Technical Information of China (English)

    YU Jie; YANG Haiquan; TAN Ming; ZHANG Guoning

    2006-01-01

    Extraction of buildings from LIDAR data has been an active research field in recent years. A scheme for building detection and reconstruction from LIDAR data is presented with an object-oriented method which is based on the buildings' semantic rules. Two key steps are discussed: how to group the discrete LIDAR points into single objects and how to establish the buildings' semantic rules. In the end, the buildings are reconstructed in 3D form and three common parametric building models (flat, gabled, hipped) are implemented.

  10. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    Science.gov (United States)

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  11. (Nontranslational medicine: RNA-based therapeutics in bacteria

    Directory of Open Access Journals (Sweden)

    Adam M Dinan

    2013-11-01

    Full Text Available The rise and spread of antibiotic resistance is among the most severe challenges facing modern medicine. Despite this fact, attempts to develop novel classes of antibiotic have been largely unsuccessful. The traditional mechanisms by which antibiotics work are subject to relatively rapid bacterial resistance via mutation, and hence have a limited period of efficacy. One promising strategy to ameliorate this problem is to shift from the use of chemical compounds targeting protein structures and processes to a new era of RNA-based therapeutics. RNA-mediated regulation (riboregulation has evolved naturally in bacteria and is therefore a highly efficient means by which gene expression can be manipulated. Here, we describe recent advances towards the development of effective anti-bacterial therapies, which operate through various strategies centred on RNA. Significant challenges facing the field, including the identification of suitable molecular targets and delivery strategies, will also be considered.

  12. 辣椒叶片总RNA快速提取%Rapid Extraction of Total RNA from Capsicum annuum Leaves

    Institute of Scientific and Technical Information of China (English)

    李小霞; 肖仲久; 宋培勇; 周逊; 谢语

    2011-01-01

    Trizol extraction method was modified to extract total RNA from Capsicum annuum leaves. The result of agar gel electrophoresis, ultraviolet ray photometer and RT-PCR showed that the total RNA obtained by modified Trizol method was of high quality, and suitable for downstream applications.%采用改良的Trizol法对辣椒(Capsicum annuum)叶片的总RNA进行了提取,利用琼脂糖凝胶电泳、紫外分光光度法、RT-PCR进行RNA纯度、完整性检测.结果表明,Trizol法提取可获得较高质量的辣椒叶片总RNA,能满足后续的研究需要.

  13. Total RNA extraction of Cryptococcus neoformans%新生隐球菌总 RNA 抽提方法的实验研究

    Institute of Scientific and Technical Information of China (English)

    徐赤宇; 刘翠杰; 吴建华; 仇芸; 赵瑾; 朱红梅; 温海

    2012-01-01

    目的 探讨快速获取高质量的新生隐球菌总RNA的实验方法.方法 选取新生隐球菌的荚膜株、荚膜缺陷株,分别设计采用4种方法提取总RNA:酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法.用紫外线分光光度计测量其OD260、0D280的值,并且进行琼脂糖凝胶电泳,同时应用定量PCR法鉴定RNA质量.结果 酸洗玻璃珠法、液氮研磨法、异硫氰酸胍一步法、冷酸洗玻璃珠联合Yeast RNA kit法的RNA产量分别为0.2μg/105细胞、0.4.μg/105细胞、0.1 μg/105细胞、0.6 μg/105细胞.结论 冷酸洗玻璃珠联合Yeast RNA kit法提取的RNA均一性和完整性最好,是简便、快捷地提取具有荚膜和细胞壁双重屏障的新生隐球菌RNA的理想方法.%Objective A quick approach to extract high quality total RNA from Cryptococcus neoformans plays a vital role in researches on the molecular detection and disease-related gene expression of high pathogenic Cryptococcus neoformans . Method Cryptoccocus neoformans strain and Cryptoccocus neoformans capsule deficient strain were used in the research. Four methods were designed to perform the extraction: acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach. RNA quality is determined by measurement of OD260 and OD280 with UV spectrophotometer, by agarose gel electrophoresis and by quantitative PCR. Result RNA yield of the four approaches, namely, acid washed glass beads approach, liquid nitrogen approach, guanidinium isothiocyanate one step approach and acid washed glass beads associated with Yeast RNA kit approach, was 0. 2 μg/10 cells, 0.4 μg/10 cells, 0. 1 μg/10 cells and 0. 6 μg/105 cells respectively. Conclusion RNA which gained by the approach of acid washed glass beads associated with Yeast RNA kit has the best homogeneity and integrity. Thus, this

  14. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    Science.gov (United States)

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. PMID:27321701

  15. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

    Science.gov (United States)

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

    2015-12-29

    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

  16. FEATURE EXTRACTION FOR EMG BASED PROSTHESES CONTROL

    Directory of Open Access Journals (Sweden)

    R. Aishwarya

    2013-01-01

    Full Text Available The control of prosthetic limb would be more effective if it is based on Surface Electromyogram (SEMG signals from remnant muscles. The analysis of SEMG signals depend on a number of factors, such as amplitude as well as time- and frequency-domain properties. Time series analysis using Auto Regressive (AR model and Mean frequency which is tolerant to white Gaussian noise are used as feature extraction techniques. EMG Histogram is used as another feature vector that was seen to give more distinct classification. The work was done with SEMG dataset obtained from the NINAPRO DATABASE, a resource for bio robotics community. Eight classes of hand movements hand open, hand close, Wrist extension, Wrist flexion, Pointing index, Ulnar deviation, Thumbs up, Thumb opposite to little finger are taken into consideration and feature vectors are extracted. The feature vectors can be given to an artificial neural network for further classification in controlling the prosthetic arm which is not dealt in this paper.

  17. Radiorestoration of the tumor tissues of Scorzonera exposed to Co60 gamma-ray through 3 cytokinins contained in the extracts and RNA yeast

    International Nuclear Information System (INIS)

    The 3 natural cytokinins contained both in the extracts and RNA of yeast and the tumor tissues cultivated in vitro, elicit radiorestorative properties from the crown-gall tissues of Scorsonera submitted to Co60 gamma-rays. However the cytokinins produce no simulating effect on the non irradiated tissues that are nontheless highly stimulated by yeast extract and their RNA

  18. Context Based Word Sense Extraction in Text

    Directory of Open Access Journals (Sweden)

    Ranjeetsingh S.Suryawanshi

    2011-11-01

    Full Text Available In the era of modern e-document technology, everyone using computerized document for their purpose. Due to huge amount of text document available in the form of pdf, doc, txt, html, and xml user may confuse about reading sense of these entire documents, if same word interpret different sense. Word sense has always been an important problem in information retrieval and extraction, as well as, text mining, because machines don’t have that much intelligence as compared to human to sense word in particular context. User want to determine which sense of a word is used in a given context. Word is usage-based, and part of it can be created automatically from an electronic dictionary. This paper describes word sense as expressed by its WordNet synsets, arranged according to their relevance and their context are expressed by means of word association

  19. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    OpenAIRE

    Vasila Packeer Mohamed; Yumi Z. H-Y. Hashim; A. Amid; M. Mel

    2014-01-01

    ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR) is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively l...

  20. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

    Directory of Open Access Journals (Sweden)

    Tanupat Mornkham

    2013-04-01

    Full Text Available Jerusalem artichoke (Helianthus tuberosus L. is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011 yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

  1. Co-LncRNA: investigating the lncRNA combinatorial effects in GO annotations and KEGG pathways based on human RNA-Seq data.

    Science.gov (United States)

    Zhao, Zheng; Bai, Jing; Wu, Aiwei; Wang, Yuan; Zhang, Jinwen; Wang, Zishan; Li, Yongsheng; Xu, Juan; Li, Xia

    2015-01-01

    Long non-coding RNAs (lncRNAs) are emerging as key regulators of diverse biological processes and diseases. However, the combinatorial effects of these molecules in a specific biological function are poorly understood. Identifying co-expressed protein-coding genes of lncRNAs would provide ample insight into lncRNA functions. To facilitate such an effort, we have developed Co-LncRNA, which is a web-based computational tool that allows users to identify GO annotations and KEGG pathways that may be affected by co-expressed protein-coding genes of a single or multiple lncRNAs. LncRNA co-expressed protein-coding genes were first identified in publicly available human RNA-Seq datasets, including 241 datasets across 6560 total individuals representing 28 tissue types/cell lines. Then, the lncRNA combinatorial effects in a given GO annotations or KEGG pathways are taken into account by the simultaneous analysis of multiple lncRNAs in user-selected individual or multiple datasets, which is realized by enrichment analysis. In addition, this software provides a graphical overview of pathways that are modulated by lncRNAs, as well as a specific tool to display the relevant networks between lncRNAs and their co-expressed protein-coding genes. Co-LncRNA also supports users in uploading their own lncRNA and protein-coding gene expression profiles to investigate the lncRNA combinatorial effects. It will be continuously updated with more human RNA-Seq datasets on an annual basis. Taken together, Co-LncRNA provides a web-based application for investigating lncRNA combinatorial effects, which could shed light on their biological roles and could be a valuable resource for this community. Database URL: http://www.bio-bigdata.com/Co-LncRNA/.

  2. Fingerprint Feature Extraction Based on Macroscopic Curvature

    Institute of Scientific and Technical Information of China (English)

    Zhang Xiong; He Gui-ming; Zhang Yun

    2003-01-01

    In the Automatic Fingerprint Identification System (AFIS), extracting the feature of fingerprint is very important. The local curvature of ridges of fingerprint is irregular, so people have the barrier to effectively extract the fingerprint curve features to describe fingerprint. This article proposes a novel algorithm; it embraces information of few nearby fingerprint ridges to extract a new characteristic which can describe the curvature feature of fingerprint. Experimental results show the algorithm is feasible, and the characteristics extracted by it can clearly show the inner macroscopic curve properties of fingerprint. The result also shows that this kind of characteristic is robust to noise and pollution.

  3. Fingerprint Feature Extraction Based on Macroscopic Curvature

    Institute of Scientific and Technical Information of China (English)

    Zhang; Xiong; He; Gui-Ming; 等

    2003-01-01

    In the Automatic Fingerprint Identification System(AFIS), extracting the feature of fingerprint is very important. The local curvature of ridges of fingerprint is irregular, so people have the barrier to effectively extract the fingerprint curve features to describe fingerprint. This article proposes a novel algorithm; it embraces information of few nearby fingerprint ridges to extract a new characterstic which can describe the curvature feature of fingerprint. Experimental results show the algorithm is feasible, and the characteristics extracted by it can clearly show the inner macroscopic curve properties of fingerprint. The result also shows that this kind of characteristic is robust to noise and pollution.

  4. Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J

    1991-01-01

    We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T...... organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two...

  5. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander

    2010-12-01

    Full Text Available Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR assays. This single-tube quality control (QC assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

  6. Optimization-based Method for Automated Road Network Extraction

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, D

    2001-09-18

    Automated road information extraction has significant applicability in transportation. It provides a means for creating, maintaining, and updating transportation network databases that are needed for purposes ranging from traffic management to automated vehicle navigation and guidance. This paper is to review literature on the subject of road extraction and to describe a study of an optimization-based method for automated road network extraction.

  7. Selective electromembrane extraction based on isoelectric point

    DEFF Research Database (Denmark)

    Huang, Chuixiu; Gjelstad, Astrid; Pedersen-Bjergaard, Stig

    2015-01-01

    , and the target remained in the acceptor solution. The acceptor solution pH, the SLM composition, the extraction voltage, and the extraction time during the clean-up process (step #2) were important factors influencing the separation performance. An acceptor solution pH of 5.25 for the clean-up process slightly...

  8. Idioms-based Business Rule Extraction

    NARCIS (Netherlands)

    Smit, R

    2011-01-01

    This thesis studies the extraction of embedded business rules, using the idioms of the used framework to identify them. Embedded business rules exist as source code in the software system and knowledge about them may get lost. Extraction of those business rules could make them accessible and managea

  9. An optmized protocol for simultaneous extraction of DNA and RNA from soils Protocolo otimizado para extração simultanea de DNA e RNA de solo

    Directory of Open Access Journals (Sweden)

    Rodrigo Costa

    2004-09-01

    Full Text Available In this work we report an optimized protocol for simultaneous extraction of DNA and RNA from soil matrices. Treatment of soil matrices with ethanol followed by bead-beating worked as a successful strategy to lyse the cells without considerable degradation of nucleic acids, resulting in DNA and RNA of good yield and integrity. The reverse transcribed RNA could be amplified with primers targeting a glutamine synthetase (glnA gene fragment. From both DNA and cDNA, 16S rDNA fragments were amplified and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE. The method was applied to soil and rhizosphere (strawberry and oilseed rape samples. Two other protocols for the extraction of nucleic acids from soil were applied to the same set of samples in order to compare the methods in terms of efficiency and reproducibility. The DGGE profiles indicated no relevant differences between the patterns obtained. The method described here is suitable for rapid processing of many samples and therefore appropriate for ecological studies.Nesse trabalho descrevemos um protocolo otimizado para extração simultânea de DNA e RNA de solo. O tratamento das amostras de solo com etanol e posterior agitação com partículas foi uma estratégia bem sucedida para lise das células sem degradação significativa dos ácidos nucléicos, resultando em bom rendimento de DNA e RNA íntegros. O RNA transcrito pode ser amplificado com iniciadores com alvo no fragmento do gene da glutamina sintetase (glnA. Os fragmentos 16S rDNA, tanto do DNA como do cDNA, foram amplificados e analisados por DGGE. O método foi aplicado para amostras de solo e rizosfera (morango e canola. Dois outros protocolos para extração de ácidos nucléicos de solo foram aplicados para o mesmo lote de amostras, de forma a comparar os métodos quanto à eficiência e reprodutibilidade. Os perfis de DGGE mostraram não haver diferença relevante nos padrões obtidos. O método descrito é apropriado para

  10. Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology

    Directory of Open Access Journals (Sweden)

    McKiernan PJ

    2013-10-01

    Full Text Available Paul J McKiernan,2 Orla Cunninghamm,1,2 Catherine M Greenem,2 Sally-Ann Cryan1,31School of Pharmacy, Royal College of Surgeons in Ireland, 2Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Beaumont Hospital, 3Trinity Centre for Bioengineering, Trinity College Dublin, Dublin, IrelandAbstract: Cystic fibrosis (CF is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs are endogenous small RNAs which act on messenger (mRNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1 expression (a validated miR-126 target was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P ratios resulted in significant knockdown of

  11. A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

    Science.gov (United States)

    Alejska, Magdalena; Figlerowicz, Magdalena; Malinowska, Nelli; Urbanowicz, Anna; Figlerowicz, Marek

    2005-07-07

    At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

  12. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    Science.gov (United States)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  13. Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA

    International Nuclear Information System (INIS)

    Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11-15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3'-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO-NH2) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3'-terminal ribose. Comparison of the 500-MHz 1H NMR spectra of native and 3'-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (G1 . C120) and its adjacent base pair (G2 . U119) on the basis of their proximity to the 3'-terminal spin-label. From that starting point, we have then identified (G . C, A . U, or G . U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE's

  14. Predicting Large RNA-Like Topologies by a Knowledge-Based Clustering Approach.

    Science.gov (United States)

    Baba, Naoto; Elmetwaly, Shereef; Kim, Namhee; Schlick, Tamar

    2016-02-27

    An analysis and expansion of our resource for classifying, predicting, and designing RNA structures, RAG (RNA-As-Graphs), is presented, with the goal of understanding features of RNA-like and non-RNA-like motifs and exploiting this information for RNA design. RAG was first reported in 2004 for cataloging RNA secondary structure motifs using graph representations. In 2011, the RAG resource was updated with the increased availability of RNA structures and was improved by utilities for analyzing RNA structures, including substructuring and search tools. We also classified RNA structures as graphs up to 10 vertices (~200 nucleotides) into three classes: existing, RNA-like, and non-RNA-like using clustering approaches. Here, we focus on the tree graphs and evaluate the newly founded RNAs since 2011, which also support our refined predictions of RNA-like motifs. We expand the RAG resource for large tree graphs up to 13 vertices (~260 nucleotides), thereby cataloging more than 10 times as many secondary structures. We apply clustering algorithms based on features of RNA secondary structures translated from known tertiary structures to suggest which hypothetical large RNA motifs can be considered "RNA-like". The results by the PAM (Partitioning Around Medoids) approach, in particular, reveal good accuracy, with small error for the largest cases. The RAG update here up to 13 vertices offers a useful graph-based tool for exploring RNA motifs and suggesting large RNA motifs for design. PMID:26478223

  15. RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS.

    Science.gov (United States)

    Piatkowski, Pawel; Kasprzak, Joanna M; Kumar, Deepak; Magnus, Marcin; Chojnowski, Grzegorz; Bujnicki, Janusz M

    2016-01-01

    RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at http://genesilico.pl/modernaserver/ . SimRNA is currently available to download for local use as a precompiled software package at http://genesilico.pl/software/stand-alone/simrna and as a

  16. RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS.

    Science.gov (United States)

    Piatkowski, Pawel; Kasprzak, Joanna M; Kumar, Deepak; Magnus, Marcin; Chojnowski, Grzegorz; Bujnicki, Janusz M

    2016-01-01

    RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at http://genesilico.pl/modernaserver/ . SimRNA is currently available to download for local use as a precompiled software package at http://genesilico.pl/software/stand-alone/simrna and as a

  17. Object Extraction Based on Evolutionary Morphological Processing

    Institute of Scientific and Technical Information of China (English)

    LI Bin; PAN Li

    2004-01-01

    This paper introduces a novel technique for object detection using genetic algorithms and morphological processing. The method employs a kind of object oriented structure element, which is derived by genetic algorithms. The population of morphological filters is iteratively evaluated according to a statistical performance index corresponding to object extraction ability, and evolves into an optimal structuring element using the evolution principles of genetic search. Experimental results of road extraction from high resolution satellite images are presented to illustrate the merit and feasibility of the proposed method.

  18. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system.

    Science.gov (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith

    2011-01-01

    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  19. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    Directory of Open Access Journals (Sweden)

    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

  20. Synergistic extraction of uranium from Korean black shale ore leach liquors using amine with phosphorous based extractant systems

    International Nuclear Information System (INIS)

    Synergistic extraction of uranium using amine combined with phosphorous based extractant systems was described. The present study focused on the continuous extraction processing of uranium to form precipitation under higher pH conditions and higher aqueous phase ratios. To address this, synergistic extraction studies were carried out with P-based extractants as synergists and investigations were done with better pairs with an amine-based extractant system. Finally, all of the developed synergistic extraction methodologies were compared with each other. This showed that Alamine 336 and D2EHPA was the best pair for uranium extraction, offering as much separation as possible from other associated metals. (author)

  1. A Novel Microfluidic Device for Fully Automated Extraction of RNA from Cell Cultures Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Obtaining high quality, intact RNA from cells is an ubiquitous need in the pursuit of space biology. Our overall objective is to develop and commercialize a...

  2. A Novel Microfluidic Device for Fully Automated Extraction of RNA from Cell Cultures Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Differential gene expression by RNA profiling is a universal and critical step in space biology experiments, which seek to link specific molecular events with...

  3. RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment.

    Science.gov (United States)

    Suzuki, Yuji; Mae, Tadahiko; Makino, Amane

    2008-07-01

    High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction.

  4. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

    OpenAIRE

    Coombs, N J; Gough, A C; Primrose, J N

    1999-01-01

    Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-1...

  5. A Suitable Method of RNA Extraction from Different Oil Palm Tissues%一种适合油棕不同组织 RNA 提取的方法

    Institute of Scientific and Technical Information of China (English)

    李静; 王永; 杨耀东; 雷新涛; 肖勇; 夏薇

    2014-01-01

    High quality of RNA is the important prerequisite and guarantee to basic research related to oil palm fruit development in molecular biology. RNA extracting effects of both Trizol reagents and MRIP (Methods for RNA Isolation from Palms) method developed by our laboratory previously were compared among four oil palm tissues, i.e. mature mesocarp, endosperm, embryo and germinated plumule. MRIP can get good quality RNA.The RNA content of plumule is as high as 2 139.0 ng/μL, while that of endosperm is only 1 083.6 ng/μL. MRIP is generally better than Trizol method in respect of the integrality, content and purity of extracted RNA, which Suitable for oil palm and palm plants RNA extraction.%获得高质量的 RNA 是开展油棕果实发育相关分子生物学基础研究的重要前提和保障。以油棕成熟果肉、胚乳、胚和胚芽4种组织为材料,分别采用 Invitrogen 公司的 Trizol 试剂和 MRIP 法提取总 RNA,并比较其提取效果。结果表明: MRIP 法提取的 RNA 质量较好,其中胚芽中的总 RNA 提取量高达2139.0 ng/μL,而胚乳中的含量相比较低,仅为1083.6 ng/μL; MRIP 法的总体提取效果优于 Trizol 试剂,包括 RNA 完整性、含量、纯度等,适用于油棕及棕榈科植物总 RNA 提取。

  6. Efficient Fingerprint Matching Based Upon Minutiae Extraction

    OpenAIRE

    Chiranjeeb Roy Chowdhury; Banani Saha

    2015-01-01

    Fingerprints are one of the oldest and most widely used biometric security measures. Rapid advances in Computer Science and digital Image Processing have made it possible to design various Automatic Fingerprint Identification Systems (AFIS) which can compare certain features of an input fingerprint image with a series of template images stored in a database and find a match. This paper deals with the extraction of certain specific features from a fingerprint, called minutiae. Since low qualit...

  7. Nanomedicine Meets microRNA: Current Advances in RNA-Based Nanotherapies for Atherosclerosis.

    Science.gov (United States)

    Gadde, Suresh; Rayner, Katey J

    2016-09-01

    Cardiovascular disease (CVD) accounts for almost half of all deaths worldwide and has now surpassed infectious disease as the leading cause of death and disability in developing countries. At present, therapies such as low-density lipoprotein-lowering statins and antihypertensive drugs have begun to bend the morality curve for coronary artery disease (CAD); yet, as we come to appreciate the more complex pathophysiological processes in the vessel wall, there is an opportunity to fine-tune therapies to more directly target mechanisms that drive CAD. MicroRNAs (miRNAs) have been identified that control vascular cell homeostasis,(1-3) lipoprotein metabolism,(4-9) and inflammatory cell function.(10) Despite the importance of these miRNAs in driving atherosclerosis and vascular dysfunction, therapeutic modulation of miRNAs in a cell- and context-specific manner has been a challenge. In this review, we summarize the emergence of miRNA-based therapies as an approach to treat CAD by specifically targeting the pathways leading to the disease. We focus on the latest development of nanoparticles (NPs) as a means to specifically target the vessel wall and what the future of these nanomedicines may hold for the treatment of CAD. PMID:27559146

  8. An Improved TRIzol Method to Extract Total RNA from Skin Tissue of Rana dybowskii%改良TRIzol法高效提取东北林蛙皮肤总RNA

    Institute of Scientific and Technical Information of China (English)

    王丹丹; 肖向红; 吴立舒; 柴龙会; 张晶钰; 张建平

    2012-01-01

    Our objective was to develop a method for extracting high quality total RNA from amphibian skin tissue.We extracted total RNA from the skin of Dybowski's frog using a modified traditional TRIzol method.We assayed the purity and yield of the RNA by UV spectrophotometer.We examined the integrity of the extracted RNA by 1.1%agarose gel electrophoresis.The A260/A280 ratios of RNA extracted by our modified method ranged from 1.8 to 2.1,and the A260/A230 ratios were greater than 2.Agarose gel electrophoresis showed clear 28s rRNA and 18s rRNA bands,and the brightness of 28s rRNA bands was approximately twice the 18s rRNA.We conclude that the purity and integrity of total RNA extracted by our improved TRIzol method were higher than that extracted by the traditional TRIzol method:the total RNA had fewer impurities and the yields were larger.The modified method was easier to implement and can be used in relevant molecular biology tests.%目的:建立一种从两栖动物皮肤组织中提取高质量总RNA的方法。方法:改良传统TRIzol法,提取皮肤总RNA,紫外分光光度计测定RNA纯度和得率,1.1%琼脂糖凝胶电泳检测其完整性。结果:改良方法提取的总RNA,其A260/A280值在1.8~2.1之间,A260/A230大于2;琼脂糖凝胶电泳显示清晰的28s rRNA和18s rRNA条带,且28s rRNA条带亮度约为18s rRNA的2倍。结论:改良TRIzol法提取的总RNA纯度高、完整性好、杂质少、得率大,易于操作掌握,可以用于相应的分子生物学试验。

  9. RNA interference-based nanosystems for inflammatory bowel disease therapy

    Directory of Open Access Journals (Sweden)

    Guo J

    2016-10-01

    Full Text Available Jian Guo,1 Xiaojing Jiang,1 Shuangying Gui1,2 1Department of Pharmaceutics, College of Pharmacy, Anhui University of Chinese Medicine, 2Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei, Anhui, People’s Republic of China Abstract: Inflammatory bowel disease (IBD, which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA and microRNA (miRNA. However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. Keywords: RNA interference, siRNA, miRNA, nanoparticles, inflammatory bowel disease, target therapy

  10. Dwt - Based Feature Extraction from ecg Signal

    Directory of Open Access Journals (Sweden)

    V.K.Srivastava

    2013-01-01

    Full Text Available Electrocardiogram is used to measure the rate and regularity of heartbeats to detect any irregularity to the heart. An ECG translates the heart electrical activity into wave-line on paper or screen. For the feature extraction and classification task we will be using discrete wavelet transform (DWT as wavelet transform is a two-dimensional timescale processing method, so it is suitable for the non-stationary ECG signals(due to adequate scale values and shifting in time. Then the data will be analyzed and classified using neuro-fuzzy which is a hybrid of artificial neural networks and fuzzy logic.

  11. Connection Skeleton Extraction Based on Contour Connectedness

    Institute of Scientific and Technical Information of China (English)

    CHEN Mang; LIU Yun-cai

    2008-01-01

    A stable skeleton is very important to some applications such as vehicle navigation, object represent and pattern recognition. The connection skeleton is just one that not only can be computed stably but also can figure the connectivity structure of contour. A new method named continuous connectivity detection and a new model named approximate regular polygon (ARP) were proposed for connection skeleton extraction. Both the method and the model were tested by the real maps of road network including flyovers, interchanges and other common object contours. Satisfactory results were obtained.

  12. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    Science.gov (United States)

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

  13. A fast quantum mechanics based contour extraction algorithm

    Science.gov (United States)

    Lan, Tian; Sun, Yangguang; Ding, Mingyue

    2009-02-01

    A fast algorithm was proposed to decrease the computational cost of the contour extraction approach based on quantum mechanics. The contour extraction approach based on quantum mechanics is a novel method proposed recently by us, which will be presented on the same conference by another paper of us titled "a statistical approach to contour extraction based on quantum mechanics". In our approach, contour extraction was modeled as the locus of a moving particle described by quantum mechanics, which is obtained by the most probable locus of the particle simulated in a large number of iterations. In quantum mechanics, the probability that a particle appears at a point is equivalent to the square amplitude of the wave function. Furthermore, the expression of the wave function can be derived from digital images, making the probability of the locus of a particle available. We employed the Markov Chain Monte Carlo (MCMC) method to estimate the square amplitude of the wave function. Finally, our fast quantum mechanics based contour extraction algorithm (referred as our fast algorithm hereafter) was evaluated by a number of different images including synthetic and medical images. It was demonstrated that our fast algorithm can achieve significant improvements in accuracy and robustness compared with the well-known state-of-the-art contour extraction techniques and dramatic reduction of time complexity compared to the statistical approach to contour extraction based on quantum mechanics.

  14. An Effective History-based Background Extraction System

    Directory of Open Access Journals (Sweden)

    Fatimah Khalid

    2012-01-01

    Full Text Available Problem statement: In many visions-based surveillance systems, the first step is accomplished by detecting moving objects resulted from subtraction of the current captured frame from the extracted background. So, the results of these systems mainly depend on the accuracy of the background image. Approach: In this study, a proposed background extraction system is presented to model the background using a simple method, to initialize the model, to extract the moving objects and to construct the final background. Our model saves the history of each pixel separately. It uses the saved information to extract the background using a probability-based method. It updates the history of the pixel consequently and according to the value of that pixel in the current captured image. Results: Results of the experiments certify that not only the quality of the final extracted background is the best between four recently re-implemented methods, but also the time consumption of the extraction is acceptable. Conclusion: Since History-based methods use temporal information extracted from the several previous frames, they are less sensitive to noise and sudden changes for extracting the background image.

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

    DEFF Research Database (Denmark)

    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....

  16. Efficient Fingerprint Matching Based Upon Minutiae Extraction

    Directory of Open Access Journals (Sweden)

    Chiranjeeb Roy Chowdhury

    2015-12-01

    Full Text Available Fingerprints are one of the oldest and most widely used biometric security measures. Rapid advances in Computer Science and digital Image Processing have made it possible to design various Automatic Fingerprint Identification Systems (AFIS which can compare certain features of an input fingerprint image with a series of template images stored in a database and find a match. This paper deals with the extraction of certain specific features from a fingerprint, called minutiae. Since low quality images tend to generate multiple false minutiae, a method has been included to detect and remove such false minutiae. Fingerprint matching is performed by matching the number and type of the minutiae. The false minutiae removal process helps to reduce the computational complexity and improve the accuracy of the match.

  17. Driver’s Fatigue Detection Based on Yawning Extraction

    Directory of Open Access Journals (Sweden)

    Nawal Alioua

    2014-01-01

    a real danger on road since it reduces driver capacity to react and analyze information. In this paper we propose an efficient and nonintrusive system for monitoring driver fatigue using yawning extraction. The proposed scheme uses face extraction based support vector machine (SVM and a new approach for mouth detection, based on circular Hough transform (CHT, applied on mouth extracted regions. Our system does not require any training data at any step or special cameras. Some experimental results showing system performance are reported. These experiments are applied over real video sequences acquired by low cost web camera and recorded in various lighting conditions.

  18. A Robust Digital Watermark Extracting Method Based on Neural Network

    Institute of Scientific and Technical Information of China (English)

    GUOLihua; YANGShutang; LIJianhua

    2003-01-01

    Since watermark removal software, such as StirMark, has succeeded in washing watermarks away for most of the known watermarking systems, it is necessary to improve the robustness of watermarking systems. A watermark extracting method based on the error Back propagation (BP) neural network is presented in this paper, which can efficiently improve the robustness of watermarking systems. Experiments show that even if the watermarking systems are attacked by the StirMark software, the extracting method based on neural network can still efficiently extract the whole watermark information.

  19. Extraction of Textual Causal Relationships based on Natural Language Processing

    Directory of Open Access Journals (Sweden)

    Sepideh Jamshidi-Nejad

    2015-11-01

    Full Text Available Natural language processing is a highly important subcategory in the wide area of artificial intelligence. Employing appropriate computational algorithms on sophisticated linguistic operations is the aim of natural language processing to extract and create computational theories from languages. In order to achieve this goal, the knowledge of linguists is needed in addition to computer science. In the field of linguistics, the syntactic and semantic relation of words and phrases and the extraction of causation is very significant which the latter is an information retrieval challenge. Recently, there is an increased attention towards the automatic extraction of causation from textual data sets. Although, previous research extracted the casual relations from uninterrupted data sets by using knowledge-based inference technologies and manual coding. Recently, finding comprehensive approaches for detection and extractions of causal arguments is a research area in the field of natural language processing.In this paper, a three-stepped approach is established through which, the position of words with syntax trees is obtained by extracting causation from causal and non-causal sentences of Web text. The arguments of events were extracted according to the dependency tree of phrases implemented by Python packages. Then potential causal relations were extracted by the extraction of specific nodes of the tree. In the final step, a statistical model is introduced for measuring the potential causal relations. Experimental results and evaluations with Recall, Precision and F-measure metrics show the accuracy and efficiency of the suggested model.

  20. RNA-based networks: using RNA aptamers and ribozymes as synthetic genetic devices.

    Science.gov (United States)

    Weigand, Julia E; Wittmann, Alexander; Suess, Beatrix

    2012-01-01

    Within the last few years, a set of synthetic riboswitches has been engineered, which expands the toolbox of genetic regulatory devices. Small molecule binding aptamers have been used for the design of such riboswitches by insertion into untranslated regions of mRNAs, exploiting the fact that upon ligand binding the RNA structure interferes either with translation initiation or pre-mRNA splicing in yeast. In combination with self-cleaving ribozymes, aptamers have been used to modulate RNA stability. In this chapter, we discuss the applicability of different aptamers, ways to identify novel genetic devices, the pros and cons of various insertion sites and the application of allosteric ribozymes. Our expertise help to apply synthetic riboswitches to engineer complex genetic circuits. PMID:22083741

  1. A simple procedure for routine RNA extraction and miRNA array analyses from a single thyroid in vivo fine needle aspirate

    DEFF Research Database (Denmark)

    Rossing, Maria; Kaczkowski, Bogumil; Futoma-Kazmierczak, Ewa;

    2010-01-01

    microRNA (miRNA) expression profiling and classification of tissue obtained from fine-needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules.......microRNA (miRNA) expression profiling and classification of tissue obtained from fine-needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules....

  2. End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

    Science.gov (United States)

    Derr, Alan; Yang, Chaoxing; Zilionis, Rapolas; Sergushichev, Alexey; Blodgett, David M.; Redick, Sambra; Bortell, Rita; Luban, Jeremy; Harlan, David M.; Kadener, Sebastian; Greiner, Dale L.; Klein, Allon; Artyomov, Maxim N.

    2016-01-01

    RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3′-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct β-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing. PMID:27470110

  3. [Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii].

    Science.gov (United States)

    Wei, Rong-Chang; Zhao, Huan; Ma, Xiao-Jun; Mi, Ke; Mo, Chang-Ming; Pan, Li-Mei; Bai, Long-Hua; Tang, Qi

    2014-01-01

    Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

  4. RNA interference-based nanosystems for inflammatory bowel disease therapy

    Science.gov (United States)

    Guo, Jian; Jiang, Xiaojing; Gui, Shuangying

    2016-01-01

    Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi) precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA) and microRNA (miRNA). However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. PMID:27789943

  5. Study on Methods of Total RNA Extraction from Cotyledon of Chinese Chestnut%板栗子叶总RNA提取方法研究

    Institute of Scientific and Technical Information of China (English)

    王滨蔚; 车鹏燕; 何承忠; 王猛; 石卓功

    2012-01-01

    为了获得板栗子叶总RNA提取的理想方法,为后续的分子生物学研究打下基础,以板栗幼果子叶为材料,采用改良CTAB法、硼砂-CTAB法、酚-SDS法、皂土法和2种植物总RNA提取试剂盒共6种方法提取板栗子叶总RNA.结果表明,除硼砂-CTAB法外,其他5种方法均能获得板栗子叶总RNA.但是,与酚-SDS法、皂土法和2种植物总RNA提取试剂盒相比较,改良CTAB法提取的板栗子叶总RNA完整性好,纯度高,且无DNA污染,能够满足cDNA-AFLP等分子生物学研究的要求.%In order to obtain an ideal total RNA extraction method of cotyledon in Chinese chestnuts for providing foundation for molecular biology research, total RNA of cotyledon excised from young fruit of Chinese chestnut were extracted with 6 methods, including improved CTAB method, borax-CTAB method, phenol-SDS method, bentonite method and 2 RNA extraction kits. The results showed that total RNA was obtained with 5 methods but borax-CTAB method. However, the improved CTAB method was better than phenol-SDS method, bentonite method and two RNA extraction kits. The total RNA extracted with improved CTAB method had high integrity, high purity and no DNA pollution, and would be suitable for related molecular researches, such as cDNA-AFLP markers.

  6. Behavior Based Social Dimensions Extraction for Multi-Label Classification.

    Directory of Open Access Journals (Sweden)

    Le Li

    Full Text Available Classification based on social dimensions is commonly used to handle the multi-label classification task in heterogeneous networks. However, traditional methods, which mostly rely on the community detection algorithms to extract the latent social dimensions, produce unsatisfactory performance when community detection algorithms fail. In this paper, we propose a novel behavior based social dimensions extraction method to improve the classification performance in multi-label heterogeneous networks. In our method, nodes' behavior features, instead of community memberships, are used to extract social dimensions. By introducing Latent Dirichlet Allocation (LDA to model the network generation process, nodes' connection behaviors with different communities can be extracted accurately, which are applied as latent social dimensions for classification. Experiments on various public datasets reveal that the proposed method can obtain satisfactory classification results in comparison to other state-of-the-art methods on smaller social dimensions.

  7. Behavior Based Social Dimensions Extraction for Multi-Label Classification.

    Science.gov (United States)

    Li, Le; Xu, Junyi; Xiao, Weidong; Ge, Bin

    2016-01-01

    Classification based on social dimensions is commonly used to handle the multi-label classification task in heterogeneous networks. However, traditional methods, which mostly rely on the community detection algorithms to extract the latent social dimensions, produce unsatisfactory performance when community detection algorithms fail. In this paper, we propose a novel behavior based social dimensions extraction method to improve the classification performance in multi-label heterogeneous networks. In our method, nodes' behavior features, instead of community memberships, are used to extract social dimensions. By introducing Latent Dirichlet Allocation (LDA) to model the network generation process, nodes' connection behaviors with different communities can be extracted accurately, which are applied as latent social dimensions for classification. Experiments on various public datasets reveal that the proposed method can obtain satisfactory classification results in comparison to other state-of-the-art methods on smaller social dimensions. PMID:27049849

  8. Behavior Based Social Dimensions Extraction for Multi-Label Classification

    Science.gov (United States)

    Li, Le; Xu, Junyi; Xiao, Weidong; Ge, Bin

    2016-01-01

    Classification based on social dimensions is commonly used to handle the multi-label classification task in heterogeneous networks. However, traditional methods, which mostly rely on the community detection algorithms to extract the latent social dimensions, produce unsatisfactory performance when community detection algorithms fail. In this paper, we propose a novel behavior based social dimensions extraction method to improve the classification performance in multi-label heterogeneous networks. In our method, nodes’ behavior features, instead of community memberships, are used to extract social dimensions. By introducing Latent Dirichlet Allocation (LDA) to model the network generation process, nodes’ connection behaviors with different communities can be extracted accurately, which are applied as latent social dimensions for classification. Experiments on various public datasets reveal that the proposed method can obtain satisfactory classification results in comparison to other state-of-the-art methods on smaller social dimensions. PMID:27049849

  9. Research on extraction of aeromagnetic weak information based on GIS

    International Nuclear Information System (INIS)

    This paper introduces the meaning of weak information extraction and discusses the theoretic foundation of aeromagnetic weak information extraction and expounds the principle of selected mathematic-physics methods. A kind of data visualization method--'1/4 standard deviation', which is favorable for displaying weak information, is proposed in the GIS software platform. The validity test of weak information extraction has been completed based on the GIS platform in Dongsheng area. Results indicate that the weak anomaly enhancement technique may effectively restrain the interfering noise, improve the signal-to-noise ratio and further delineate the prospective targets of sandstone uranium deposits

  10. Development of a flat membrane based device for electromembrane extraction

    DEFF Research Database (Denmark)

    Huang, Chuixiu; Eibak, Lars Erik Eng; Gjelstad, Astrid;

    2014-01-01

    In this work, a single-well electromembrane extraction (EME) device was developed based on a thin (100μm) and flat porous membrane of polypropylene supporting a liquid membrane. The new EME device was operated with a relatively large acceptor solution volume to promote a high recovery. Using...... for obtaining exhaustive extraction. 2-Nitrophenyl octyl ether was selected as the optimal organic solvent for the supported liquid membrane. From spiked acidified water samples (600μl), EME was carried out with 600μl of 20mM HCOOH as acceptor solution for 15min and with an extraction voltage of 250V. Under...

  11. Perceptual Object Extraction Based on Saliency and Clustering

    Directory of Open Access Journals (Sweden)

    Qiaorong Zhang

    2010-08-01

    Full Text Available Object-based visual attention has received an increasing interest in recent years. Perceptual object is the basic attention unit of object-based visual attention. The definition and extraction of perceptual objects is one of the key technologies in object-based visual attention computation model. A novel perceptual object definition and extraction method is proposed in this paper. Based on Gestalt theory and visual feature integration theory, perceptual object is defined using homogeneity region, salient region and edges. An improved saliency map generating algorithm is employed first. Based on the saliency map, salient edges are extracted. Then graph-based clustering algorithm is introduced to get homogeneity regions in the image. Finally an integration strategy is adopted to combine salient edges and homogeneity regions to extract perceptual objects. The proposed perceptual object extraction method has been tested on lots of natural images. Experiment results and analysis are presented in this paper also. Experiment results show that the proposed method is reasonable and valid.

  12. Extracting Moving Objects Based on Morphological Motion Filter

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This paper presents a new approach to the extraction of a moving object from video sequence. The method is based on mor phological motion filter using connected operator and a proposed new filtering criterion. The morphological motion filter aims to detect motion which is distinct from that of the background, and thereby locates independently moving physical objects in the scenes. Experi ments show that the algorithm can extract object from moving backgrounds efficiently.

  13. Feature Extraction based Face Recognition, Gender and Age Classification

    OpenAIRE

    Venugopal K R2; L M Patnaik; Ramesha K; K B Raja

    2010-01-01

    The face recognition system with large sets of training sets for personal identification normally attains good accuracy. In this paper, we proposed Feature Extraction based Face Recognition, Gender and Age Classification (FEBFRGAC) algorithm with only small training sets and it yields good results even with one image per person. This process involves three stages: Pre-processing, Feature Extraction and Classification. The geometric features of facial images like eyes, nose, mouth etc. are loc...

  14. Algebraic characterization of RNA operations for DNA-based computation

    Institute of Scientific and Technical Information of China (English)

    LI Shuchao

    2004-01-01

    Any RNA strand can be presented by a word in the language X*, where X={A,C,G,U}. By encoding A as 010, C as 000, G as 111, and U as 101, the RNA operations can be considered as the performance of concatenation, union, reverse, complement, in terms of the algebraic characterization. The concatenation and union play the roles of multiplication and addition over some algebraic structures, respectively. The rest of the operations turn out to be the homomorphisms or anti-homomorphisms of these algebraic structures. Using this technique, we find the relationship among these RNA operations.

  15. Analysis of energy-based algorithms for RNA secondary structure prediction

    OpenAIRE

    Hajiaghayi Monir; Condon Anne; Hoos Holger H

    2012-01-01

    Abstract Background RNA molecules play critical roles in the cells of organisms, including roles in gene regulation, catalysis, and synthesis of proteins. Since RNA function depends in large part on its folded structures, much effort has been invested in developing accurate methods for prediction of RNA secondary structure from the base sequence. Minimum free energy (MFE) predictions are widely used, based on nearest neighbor thermodynamic parameters of Mathews, Turner et al. or those of Andr...

  16. Phylogenetic relationships of Salmonella based on rRNA sequences

    DEFF Research Database (Denmark)

    Christensen, H.; Nordentoft, Steen; Olsen, J.E.

    1998-01-01

    To establish the phylogenetic relationships between the subspecies of Salmonella enterica (official name Salmonella choleraesuis), Salmonella bongori and related members of Enterobacteriaceae, sequence comparison of rRNA was performed by maximum-likelihood analysis. The two Salmonella species were...

  17. Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt).

    Science.gov (United States)

    Xu, Qiang; Wen, Xiaopeng; Tao, Nengguo; Hu, Zhiyong; Yue, Hailin; Deng, Xiuxin

    2006-04-01

    Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and beta-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 microg RNA g(-1) fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes.

  18. Mechanism of Action and Antiviral Activity of Benzimidazole-Based Allosteric Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase

    OpenAIRE

    Tomei, Licia; Altamura, Sergio; Bartholomew, Linda; Biroccio, Antonino; Ceccacci, Alessandra; Pacini, Laura; Narjes, Frank; Gennari, Nadia; Bisbocci, Monica; Incitti, Ilario; Orsatti, Laura; Harper, Steven; Stansfield, Ian; Rowley, Michael; De Francesco, Raffaele

    2003-01-01

    The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis...

  19. Investigation of RNA Structure by High-Throughput SHAPE-Based Probing Methods

    DEFF Research Database (Denmark)

    Poulsen, Line Dahl

    of highthroughput SHAPE-based approaches to investigate RNA structure based on novel SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection (SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA genome, and the data is used to construct a genome-wide structural...

  20. Total RNA extraction of Ginkgo biloba leaves%银杏RNA的提取

    Institute of Scientific and Technical Information of China (English)

    程水源; 陈昆松; 杜何为; 许文平

    2005-01-01

    采用CTAB法提取银杏叶的RNA,结果表明:抽提的RNA经电泳检测,可见28 S rRNA和18 S rRNA两条主带;紫外吸收值测量,A260/A280接近2.0;反转录合成的cDNA经RAPD扩增,出现清晰的条带,这些说明提取的RNA纯度高,质量好.此方法简便易行,成本也较低,适合于富含多糖、多酚植物材料的RNA提取.

  1. Fractionation and characterization of a yeast mRNA splicing extract.

    OpenAIRE

    S. C. Cheng; Abelson, J

    1986-01-01

    We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the react...

  2. Influence of blood storage time on viral RNA extraction for the detection of bovine viral diarrhea virus in persistently infected cattle

    OpenAIRE

    Liu, Yingxia; Jie CAO; Zhang, Junjie; Huang, Kai; QI, Changming

    2013-01-01

    This study aimed to determine the maximum permissible storage times of blood and serum infected with bovine viral diarrhea virus (BVDV). Viral RNA was successfully extracted from blood and serum that were stored at room temperature (RT) for 7 days and was detected by 1-step RT-PCR. The results of this study demonstrate that BVDV-infected blood can be stored at RT for 7 days and that serum can be stored for 10 days without influencing the viral RNA extraction for the detection of BVDV.

  3. A Keyword Extraction Based Model for Web Advertisement

    Science.gov (United States)

    Zhou, Ning; Wu, Jiaxin; Zhang, Shaolong

    In this paper, a keyword extraction based model is proposed to deal with web advertisement. In our model, we take web advertisement as an information retrieval problem. Web page and advertisement are firstly represented with a simple data structure which will be the source file for keyword extraction based on x 2-measure for single document. Later we get two vectors to make a retrieval process with a specific similarity function. This model is suitable for common cases of web advertisement. It supports the web page selection in view of advertisement as well as the advertisement selection for specific web page.

  4. Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Eiko E Kuramae

    Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

  5. Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review.

    Science.gov (United States)

    Andries, Oliwia; Kitada, Tasuku; Bodner, Katie; Sanders, Niek N; Weiss, Ron

    2015-02-01

    Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination.

  6. GARN: Sampling RNA 3D Structure Space with Game Theory and Knowledge-Based Scoring Strategies.

    Science.gov (United States)

    Boudard, Mélanie; Bernauer, Julie; Barth, Dominique; Cohen, Johanne; Denise, Alain

    2015-01-01

    Cellular processes involve large numbers of RNA molecules. The functions of these RNA molecules and their binding to molecular machines are highly dependent on their 3D structures. One of the key challenges in RNA structure prediction and modeling is predicting the spatial arrangement of the various structural elements of RNA. As RNA folding is generally hierarchical, methods involving coarse-grained models hold great promise for this purpose. We present here a novel coarse-grained method for sampling, based on game theory and knowledge-based potentials. This strategy, GARN (Game Algorithm for RNa sampling), is often much faster than previously described techniques and generates large sets of solutions closely resembling the native structure. GARN is thus a suitable starting point for the molecular modeling of large RNAs, particularly those with experimental constraints. GARN is available from: http://garn.lri.fr/.

  7. PULMONARY VESSEL EXTRACTION BASED ON COHERENCE DIFFUSION AND FAST MARCHING

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To accurately extract pulmonary vessels on medical images. Methods An efficient vessel segmentation framework is presented, which includes a smoothing method and a extraction algorithm. The smoothing method is based on an improved coherence diffusion approach that integrates the second-order directional differential information. It can analyze weak edges such as narrow peak or ridge-like structures. Meanwhile, an improved extraction algorithm is proposed. It is based on a fast marching algorithm where a sorted sequence array and multi-initialization technique are applied. Results The improved coherence diffusion approach can precisely preserve important oriented patterns and remove noises on the images. Experimental results on several images show that the proposed method can effectively find the location of pulmonary vessels. Conclusion The segmentation method is accurate and fast that can be a useful tool for medical imaging applications.

  8. Structure-Based Design of Dendritic Peptide Bolaamphiphiles for siRNA Delivery

    OpenAIRE

    Zeng, Hanxiang; Mark E Johnson; Oldenhuis, Nathan J.; Tiambeng, Timothy N.; Guan, Zhibin

    2015-01-01

    Development of safe and effective delivery vectors is a critical challenge for the application of RNA interference (RNAi)-based biotechnologies. In this study we show the rational design of a series of novel dendritic peptide bolaamphiphile vectors that demonstrate high efficiency for the delivery of small interfering RNA (siRNA) while exhibiting low cytotoxicity and hemolytic activity. Systematic investigation into structure–property relationships revealed an important correlation between mo...

  9. SnapShot-Seq: a method for extracting genome-wide, in vivo mRNA dynamics from a single total RNA sample.

    Directory of Open Access Journals (Sweden)

    Jesse M Gray

    Full Text Available mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

  10. Small interfering RNA-based molecular therapy of cancers

    Institute of Scientific and Technical Information of China (English)

    Wei Guo; Wangbing Chen; Wendan Yu; Wenlin Huang; Wuguo Deng

    2013-01-01

    RNA interference (RNAi) has become a gold standard for validating gene function in basic life science research and provides a promising therapeutic modality for cancer and other diseases. This mini-review focuses on the potential of smal interfering RNAs (siRNAs) in anticancer treatment, including the establishment and screening of cancer-associated siRNA libraries and their applications in anticancer drug target discovery and cancer therapy. This article also describes the current delivery approaches of siRNAs using lipids, polymers, and, in particular, gold nanoparticles to induce significant gene silencing and tumor growth regression.

  11. A Block-Based Multi-Scale Background Extraction Algorithm

    Directory of Open Access Journals (Sweden)

    Seyed H. Davarpanah

    2010-01-01

    Full Text Available Problem statement: To extract the moving objects, vision-based surveillance systems subtract the current image from a predefined background image. The efficiency of these systems mainly depends on accuracy of the extracted background image. It should be able to adapt to the changes continuously. In addition, especially in real-time applications the time complexity of this adaptation is a critical matter. Approach: In this study, to extract an adaptive background, a combination of blocking and multi-scale methods is presented. Because of being less sensitive to local movements, block-based techniques are proper to control the non-stationary objects’ movements, especially in outdoor applications. They can be useful to reduce the effect of these objects on the extracted background. We also used the blocking method to intelligently select the regions which the temporal filtering has to be applied on. In addition, an amended multi-scale algorithm is introduced. This algorithm is a hybrid algorithm, a combination of some nonparametric and parametric filters. It uses a nonparametric filter in the spatial domain to initiate two primary backgrounds. In continue two adapted two-dimensional filters will be used to extract the final background. Results: The qualitative and quantitative results of our experiments certify not only the quality of the final extracted background is acceptable, but also its time consumption is approximately half in compare to the similar methods. Conclusion: Using Multi scaling filtering and applying the filters just to some selected nonoverlapped blocks reduce the time consumption of the extracting background algorithm.

  12. Lipid-based siRNA Delivery Systems: Challenges, Promises and Solutions Along the Long Journey.

    Science.gov (United States)

    Sarisozen, Can; Salzano, Giuseppina; Torchilin, Vladimir P

    2016-01-01

    RNA interference (RNAi) is an evolutionary conserved highly specific gene-silencing mechanism initiated by small interfering RNA (siRNA) molecules. Fast-paced preclinical and clinical studies helped the siRNA technology become an efficient tool for undruggable targets in different diseases including genetic diseases, viral diseases and cancer. Despite great feature of siRNAs that can down-regulate any protein in the cells, the full potential and the success of the preclinical studies could not be translated into largely successful clinical outcomes. It has become clear that the possibility of overcoming the pitfalls for in vivo siRNA therapy fully depends on delivery systems. In this review, we start with the challenges and barriers for in vivo siRNA delivery. Then we briefly discuss the recent developments in siRNA modification technology. We specifically focused on siRNA lipidation and delivery approaches with special emphasis on the lipid based hybrid systems. Here we summarize the journey of lipid-based micelle-like nanoparticle systems that combine longevity in blood, effective cellular uptake and endosomal escape for successful siRNA delivery and discuss the multifunctional stimuli-sensitive systems based on lipids as the next generation smart systems. PMID:27033509

  13. An assay for ribonuclease activity, based on ultraviolet absorption of RNA hydrolysate, using phosphotungstic acid.

    Science.gov (United States)

    Isobe, K; Uchiyama, S

    1986-06-01

    In the method for the determination of ribonuclease activity that depends on the ultraviolet absorption of the RNA hydrolysate, the uranium reagent (25% perchloric acid solution containing 0.75% uranyl acetate) is commonly used for the efficient precipitation of the unhydrolyzed RNA. However, this reagent is always contaminated by the presence of radioactive isotopes. Radioactive uranium is one of the substances used for atomic nuclear fuel and therefore, at least in Japan, the use of uranium compounds requires permission from the government. We tried to find another efficient and non-radioactive precipitant of RNA to replace the uranium reagent, and have developed a phosphotungsten reagent (25% perchloric acid solution containing 0.75% phosphotungstic acid plus 0.6% bovine serum albumin solution) which functions as efficiently as the uranium reagent in the precipitation of RNA. A cell-free crude extract of Dictyostelium discoideum was used as the source of ribonuclease.

  14. Extraction of Micronutrient Metals from Peat-based Media Using Various Chelate-ligand and Iron-source Extractants

    Science.gov (United States)

    Objectives of the study were to determine effects of chelate-ligand (experiment 1) and iron-source (experiment 2) unbuffrered extractant solutions on substrate pH and Cu, Fe, Mn, and Zn extraction from peat-based media. Chelate-ligand extractants consisted of 5 mM solutions of ethylenediaminedisucc...

  15. VDMOSFET Model Parameter Extraction Based on Electrical and Optical Measurements

    NARCIS (Netherlands)

    Salame, C.-T.; Rizk, C.; Jelian, G.

    2001-01-01

    Lateral Device parameters for VDMOSFET (Vertical double diffused metal oxide semiconductor field effect transistor) with hexagonal cells has been extracted by an original model based on electrical and optical measurements. Using microscopic observation for the ship of the device and by C-V character

  16. Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies

    Indian Academy of Sciences (India)

    Purshotam Sharma; Abhijit Mitra; Sitansh Sharma; Harjinder Singh

    2007-09-01

    The base pairing patterns in RNA structures are more versatile and completely different as compared to DNA. We present here results of ab-initio studies of structures and interaction energies of eight selected RNA base pairs reported in literature. Interaction energies, including BSSE correction, of hydrogen added crystal geometries of base pairs have been calculated at the HF/6-31G∗∗ level. The structures and interaction energies of the base pairs in the crystal geometry are compared with those obtained after optimization of the base pairs. We find that the base pairs become more planar on full optimization. No change in the hydrogen bonding pattern is seen. It is expected that the inclusion of appropriate considerations of many of these aspects of RNA base pairing would significantly improve the accuracy of RNA secondary structure prediction.

  17. siRNA delivery with lipid-based systems

    DEFF Research Database (Denmark)

    Foged, Camilla

    2012-01-01

    in vivo, toxicity and non-specific stimulation of the immune system. To optimally design and tailor the lipidic systems for siRNA delivery, better insight is needed into the mechanisms of cell delivery. More specifically, further clarification is need regarding the nature of cell surface interactions...

  18. Effect of Green Tea Extract on Systemic Metabolic Homeostasis in Diet-Induced Obese Mice Determined via RNA-Seq Transcriptome Profiles

    Science.gov (United States)

    Choi, Ji-Young; Kim, Ye Jin; Ryu, Ri; Cho, Su-Jung; Kwon, Eun-Young; Choi, Myung-Sook

    2016-01-01

    Green tea (GT) has various health effects, including anti-obesity properties. However, the multiple molecular mechanisms of the effects have not been fully determined. The aim of this study was to elucidate the anti-obesity effects of GT via the analysis of its metabolic and transcriptional responses based on RNA-seq profiles. C57BL/6J mice were fed a normal, high-fat (60% energy as fat), or high-fat + 0.25% (w/w) GT diet for 12 weeks. The GT extract ameliorated obesity, hepatic steatosis, dyslipidemia, and insulin resistance in diet-induced obesity (DIO) mice. GT supplementation resulted in body weight gain reduction than mice fed high-fat through enhanced energy expenditure, and reduced adiposity. The transcriptome profiles of epididymal white adipose tissue (eWAT) suggested that GT augments transcriptional responses to the degradation of branched chain amino acids (BCAAs), as well as AMP-activated protein kinase (AMPK) signaling, which suggests enhanced energy homeostasis. Our findings provide some significant insights into the effects of GT for the prevention of obesity and its comorbidities. We demonstrated that the GT extract contributed to the regulation of systemic metabolic homeostasis via transcriptional responses to not only lipid and glucose metabolism, but also amino acid metabolism via BCAA degradation in the adipose tissue of DIO mice. PMID:27754422

  19. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants.

    Science.gov (United States)

    Li, Jian-Feng; Chung, Hoo Sun; Niu, Yajie; Bush, Jenifer; McCormack, Matthew; Sheen, Jen

    2013-05-01

    Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

  20. RNA structure-based ribosome recruitment: Lessons from the Dicistroviridae intergenic region IRESes

    OpenAIRE

    Pfingsten, Jennifer S; Kieft, Jeffrey S.

    2008-01-01

    In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5′ end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by m...

  1. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract

    Energy Technology Data Exchange (ETDEWEB)

    Ogg, S.C.; Anderson, P.; Wickens, M.P. (Univ. of Wisconsin, Madison (USA))

    1990-01-11

    Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intro. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, the authors determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. They demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3{prime} and 5{prime} sites as are used in C. elegans. The branch point used lies in the inserted sequences. They conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

  2. Approach to extracting hot topics based on network traffic content

    Institute of Scientific and Technical Information of China (English)

    Yadong ZHOU; Xiaohong GUAN; Qindong SUN; Wei LI; Jing TAO

    2009-01-01

    This article presents the formal definition and description of popular topics on the Internet,analyzes the relationship between popular words and topics,and finally introduces a method that uses statistics and correlation of the popular words in traffic content and network flow characteristics as input for extracting popular topics on the Internet.Based on this,this article adapts a clustering algorithm to extract popular topics and gives formalized results.The test results show that this method has an accuracy of 16.7% in extracting popular topics on the Internet.Compared with web mining and topic detection and tracking (TDT),it can provide a more suitable data source for effective recovery of Internet public opinions.

  3. Distributed Parallel Endmember Extraction of Hyperspectral Data Based on Spark

    Directory of Open Access Journals (Sweden)

    Zebin Wu

    2016-01-01

    Full Text Available Due to the increasing dimensionality and volume of remotely sensed hyperspectral data, the development of acceleration techniques for massive hyperspectral image analysis approaches is a very important challenge. Cloud computing offers many possibilities of distributed processing of hyperspectral datasets. This paper proposes a novel distributed parallel endmember extraction method based on iterative error analysis that utilizes cloud computing principles to efficiently process massive hyperspectral data. The proposed method takes advantage of technologies including MapReduce programming model, Hadoop Distributed File System (HDFS, and Apache Spark to realize distributed parallel implementation for hyperspectral endmember extraction, which significantly accelerates the computation of hyperspectral processing and provides high throughput access to large hyperspectral data. The experimental results, which are obtained by extracting endmembers of hyperspectral datasets on a cloud computing platform built on a cluster, demonstrate the effectiveness and computational efficiency of the proposed method.

  4. Overlaid caption extraction in news video based on SVM

    Science.gov (United States)

    Liu, Manman; Su, Yuting; Ji, Zhong

    2007-11-01

    Overlaid caption in news video often carries condensed semantic information which is key cues for content-based video indexing and retrieval. However, it is still a challenging work to extract caption from video because of its complex background and low resolution. In this paper, we propose an effective overlaid caption extraction approach for news video. We first scan the video key frames using a small window, and then classify the blocks into the text and non-text ones via support vector machine (SVM), with statistical features extracted from the gray level co-occurrence matrices, the LH and HL sub-bands wavelet coefficients and the orientated edge intensity ratios. Finally morphological filtering and projection profile analysis are employed to localize and refine the candidate caption regions. Experiments show its high performance on four 30-minute news video programs.

  5. Effects of Ganoderma lucidum (Higher Basidiomycetes) Extracts on the miRNA Profile and Telomerase Activity of the MCF-7 Breast Cancer Cell Line.

    Science.gov (United States)

    Gonul, Oyku; Aydin, Hikmet Hakan; Kalmis, Erbil; Kayalar, Husniye; Ozkaya, Ali Burak; Atay, Sevcan; Ak, Handan

    2015-01-01

    Ganoderma lucidum is a medicinal higher Basidiomycetes mushroom that exerts anticancer effects through several different mechanisms. This study investigated the effects of G. lucidum on the telomerase activity and microRNA (miRNA) profiles of MCF-7 cells. According to the cytotoxicity results, the G. lucidum ether extract exhibits the highest cytotoxic potency; therefore it was chosen for the subsequent telomerase activity assay and miRNA profiling. The telomerase activity observed in the cells treated with a half-maximal inhibitory concentration of G. lucidum ether extract (100 µg/mL in dimethyl sulfoxide) was 32.2% lower than that of the control cells treated with 1% dimethyl sulfoxide. Among 1066 miRNAs, the most downregulated miRNA was hsa-miR-27a* (4.469-fold), and the most upregulated miRNA was hsa-miR-1285 (10.462-fold). A database search revealed the predicted miRNAs that target the catalytic subunit of the telomerase enzyme telomerase reverse transcriptase, and only miR-3687 (upregulated 2.153-fold) and miR-1207-5p (upregulated 2.895-fold) were changed by at least 2-fold. The miRNA profile changes demonstrated in this study provide a data set regarding their effects on the pathways that regulate telomerase activity in MCF-7 breast cancer cells treated with G. lucidum. These data should aid the development of novel cancer treatment strategies.

  6. Web Entities Extraction Based on Semi-Structured Semantic Database

    Directory of Open Access Journals (Sweden)

    Fang Dong

    2013-07-01

    Full Text Available Web is the biggest source of information and contains many entities and relationships between them, extracting these data from Massive Web pages and Integrating to a Semi-Structured Data with rich semantics will be more conducive to the management and use of these web data. On this premise, a comprehensive method is proposed to perform extraction the entities and relationships from the webpages. The method consists of two steps: 1 The target Web pages which contains these entities will be found based on the combination of vision information and content of keyword, meanwhile recording the relationship between father and children target Web pages; 2 Extracting the entities with analysis of DOM tree structure of the obtained Web pages and definitions of some extraction rules. At last, the extracted data is organized into a Semi-Structured Data with special relationships. Experiments on a large number of HTML pages have showed that this method can get a high correct rate and coverage.

  7. An Improved CTAB Method for Extraction of Total RNA from Mature Leaves in Oil Sunflower%改良CTAB法提取油用向日葵成熟叶片的总RNA

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

      为从植物中提取纯度高、完整性好的RNA ,采用改良过的CTAB法提取油用向日葵改 HA89开花后12 d成熟叶片的总RNA ,并对其进行了紫外分光光度计和琼脂糖凝胶电泳分析。结果表明:得到的RNA完整性好,纯度和浓度均较高,可用于后续的RT-PCR、Northern杂交和cDNA文库的构建等分子生物学实验研究。%In order to extract RNA of high purity and good integrity from plants ,after 12 days’ flowering the total RNA from mature leaves in oil sunflower Modified HA 89 was extracted through improved CTAB meth-od .It was analyzed by UV spectrophotometer and agarose gel electrophoresis .The results showed that the structure of total RNA obtained was complete with good purity and high concentration .It could be used for subsequent experiments of molecular biology such as RT-PCR ,Northern hybridization ,cDNA library construction .

  8. Transcriptome walking: a laboratory-oriented GUI-based approach to mRNA identification from deep-sequenced data

    Directory of Open Access Journals (Sweden)

    French Andrew S

    2012-12-01

    Full Text Available Abstract Background Deep sequencing technology provides efficient and economical production of large numbers of randomly positioned, relatively short, estimates of base identities in DNA molecules. Application of this technology to mRNA samples allows rapid examination of the molecular genetic environment in individual cells or tissues, the transcriptome. However, assembly of such short sequences into complete mRNA creates a challenge that limits the usefulness of the technology, particularly when no, or limited, genomic data is available. Several approaches to this problem have been developed, but there is still no general method to rapidly obtain an mRNA sequence from deep sequence data when a specific molecule, or family of molecules, are of interest. A frequent requirement is to identify specific mRNA molecules from tissues that are being investigated by methods such as electrophysiology, immunocytology and pharmacology. To be widely useful, any approach must be relatively simple to use in the laboratory by operators without extensive statistical or bioinformatics knowledge, and with readily available hardware. Findings An approach was developed that allows de novo assembly of individual mRNA sequences in two linked stages: sequence discovery and sequence completion. Both stages rely on computer assisted, Graphical User Interface (GUI-guided, user interaction with the data, but proceed relatively efficiently once discovery is complete. The method grows a discovered sequence by repeated passes through the complete raw data in a series of steps, and is hence termed ‘transcriptome walking’. All of the operations required for transcriptome analysis are combined in one program that presents a relatively simple user interface and runs on a standard desktop, or laptop computer, but takes advantage of multi-core processors, when available. Complete mRNA sequence identifications usually require less than 24 hours. This approach has already

  9. An efficient Potato virus X -based microRNA silencing in Nicotiana benthamiana

    Science.gov (United States)

    Zhao, Jinping; Liu, Qingtao; Hu, Pu; Jia, Qi; Liu, Na; Yin, Kangquan; Cheng, Ye; Yan, Fei; Chen, Jianping; Liu, Yule

    2016-01-01

    Plant microRNAs (miRNAs) play pivotal roles in many biological processes. Although many miRNAs have been identified in various plant species, the functions of these miRNAs remain largely unknown due to the shortage of effective genetic tools to block their functional activity. Recently, miRNA target mimic (TM) technologies have been applied to perturb the activity of specific endogenous miRNA or miRNA families. We previously reported that Tobacco rattle virus (TRV)-based TM expression can successfully mediate virus-based miRNA silencing/suppression (VbMS) in plants. In this study, we show the Potato virus X (PVX)-based TM expression causes strong miRNA silencing in Nicotiana benthamiana. The PVX-based expression of short tandem target mimic (STTMs) against miR165/166 and 159 caused the corresponding phenotype in all infected plants. Thus, a PVX-based VbMS is a powerful method to study miRNA function and may be useful for high-throughput investigation of miRNA function in N. benthamiana. PMID:26837708

  10. 两种同时提取小麦根腐离蠕孢菌DNA和RNA的方法比较%Comparing Two Methods for Simultaneous Extraction of DNA and RNA for Bipolaris sorokinianum

    Institute of Scientific and Technical Information of China (English)

    王珍; 崔素萍; 崔丽琴; 左豫虎

    2011-01-01

    Obtaining sufficient quantity and high quality of RNA and DNA is the base for carrying out researches in molecular biology. DNA from Bipolaris sorokiniana was attracted by two cell wall lysis methods using lysozyme and liquid nitrogen grinding methods, respectively. Integrality and purity of nucleic acids were detected with agarose gel electrophoresis, ultraviolet absorption and PCR. Ideal results were obtained, when extracting DNA, high quality RNA was obtained at the same time, by using both methods. The results of PCR indicated that the integrality of DNA was good, The quality of DNA was similar for the two methods used, while the quality of RNA was better when extracted with lysozyme than it with liquid nitrogen grinding. The DNA extraction system of Bipolaris sorokiniana by using lysozyme method was suitable for RNA extraction, it provided a simple, safe and feasible method for extracting both DNA and RNA at the same time from Bipolaris sorokiniana.%获得足够数量和高质量的RNA和DNA是进行分子生物学研究工作的基础.以小麦根腐离蠕孢菌为原料,分别采用溶菌酶法和液氮研磨法破碎真菌细胞壁提取DNA,用琼脂糖凝胶电泳、紫外吸收及聚合酶链式反应(PCR)技术进行核酸完整性和纯度检测.结果表明:2种方法在提取DNA时都同时得到了高质量的RNA;PCR检测结果表明DNA完整性比较高,2种方法得到的DNA质量相当;溶菌酶法中得到的RNA质量优于液氮法.利用溶菌酶破壁法提取小麦根腐离蠕孢菌DNA的提取体系可用于RNA的提取,为小麦根腐离蠕孢菌核酸的DNA和RNA的同时提取提供了一种简便、安全、可行的方法.

  11. Image feature extraction based multiple ant colonies cooperation

    Science.gov (United States)

    Zhang, Zhilong; Yang, Weiping; Li, Jicheng

    2015-05-01

    This paper presents a novel image feature extraction algorithm based on multiple ant colonies cooperation. Firstly, a low resolution version of the input image is created using Gaussian pyramid algorithm, and two ant colonies are spread on the source image and low resolution image respectively. The ant colony on the low resolution image uses phase congruency as its inspiration information, while the ant colony on the source image uses gradient magnitude as its inspiration information. These two ant colonies cooperate to extract salient image features through sharing a same pheromone matrix. After the optimization process, image features are detected based on thresholding the pheromone matrix. Since gradient magnitude and phase congruency of the input image are used as inspiration information of the ant colonies, our algorithm shows higher intelligence and is capable of acquiring more complete and meaningful image features than other simpler edge detectors.

  12. Feature Extraction based Face Recognition, Gender and Age Classification

    Directory of Open Access Journals (Sweden)

    Venugopal K R

    2010-01-01

    Full Text Available The face recognition system with large sets of training sets for personal identification normally attains good accuracy. In this paper, we proposed Feature Extraction based Face Recognition, Gender and Age Classification (FEBFRGAC algorithm with only small training sets and it yields good results even with one image per person. This process involves three stages: Pre-processing, Feature Extraction and Classification. The geometric features of facial images like eyes, nose, mouth etc. are located by using Canny edge operator and face recognition is performed. Based on the texture and shape information gender and age classification is done using Posteriori Class Probability and Artificial Neural Network respectively. It is observed that the face recognition is 100%, the gender and age classification is around 98% and 94% respectively.

  13. Extracting Business Rules from COBOL: A Model-Based Tool

    OpenAIRE

    Cosentino, Valerio; Cabot, Jordi; Albert, Patrick; Bauquel, Philippe; Perronnet, Jacques

    2013-01-01

    International audience; This paper presents a Business Rule Extraction tool for COBOL systems. Starting from a COBOL program, we derive a model-based representation of the source code and we provide a set of model transformations to identify and visualize the embedded business rules. In particular, the tool facilitates the definition of an application vocabulary and the identification of relevant business variables. In addition, such variables are used as starting point to slice the code in o...

  14. CTAB法用于染病烟草植株中烟草丛顶病毒RNA的提取%Application of CTAB Method for Tobacco Bushy Top Virus RNA Extraction from Infected Tobacco Plants

    Institute of Scientific and Technical Information of China (English)

    左瑞娟; 周雨泫; 李丽娟; 董鹏; 陈海如; 李凡

    2011-01-01

    CTAB法是一种广泛用于从染病植物中提取总核酸,并用于后续相关病原物PCR检测的核酸提取法,已普遍运用于DNA病毒、植原体、细菌、真菌侵染植物的总DNA提取和病原检测.本文尝试将CTAB法用于一种由RNA病毒(烟草丛顶病毒)引起的烟草丛顶病染病组织总核酸的提取,以总核酸进行烟草丛顶病毒的RT-PCR检测,并与常规RNA病毒核酸提取法--Trizol法作比较.结果表明CTAB法和Trizol法提取的核酸用于烟草丛顶病毒RT-PCR检测时,均能扩增到目的条带,检测结果一致.在对复合侵染材料进行检测时发现,CTAB法提取的染病植物组织总核酸既包括DNA也包含RNA,有利于对DNA和RNA病原物同时进行检测;CTAB法所用试剂较Trizol法便宜,毒性小,且提取的核酸量较多、保存时间较长.因此CTAB法是一种高效、简便、经济的从烟草丛顶病染病组织中提取烟草丛顶病毒RNA的方法.%CTAB method is one of the conventional pathogenic total nucleic acids extraction ways from diseased plants, which can then be used for pathogen detection in PCR-based techniques.This method has been widely used in total nucleic acids extraction and pathogen detection from the plants infected by DNA virus, phytoplasma, bacterium and fungus.In this study, the CTAB method was tried to extract the total nucleic acids from infected tobacco plants by a RNA virus of Tobacco bushy top virus (TBTV) , and compared with Trizol method, a routine RNA virus nucleic acids extraction method, for TBTV RT-PCR detection.The RT-PCR detection results showed that TBTV expected cDNA band could be amplified from the RNA templates extracted from both two methods.The extracts include both DNA and RNA when CTAB is used to extract the total nucleic acids from the co-infected plants, and thus are beneficial to detect both DNA and RNA pathogens.The chemicals used in CTAB method are much cheaper and low toxic than Trizol reagent.Additionally, the

  15. Evolutionary Patterns of RNA-Based Duplication in Non-Mammalian Chordates

    OpenAIRE

    Ming Chen; Ming Zou; Beide Fu; Xin Li; Vibranovski, Maria D.; Xiaoni Gan; Dengqiang Wang; Wen Wang; Manyuan Long; Shunping He

    2011-01-01

    The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young re...

  16. Evaluating a Pivot-Based Approach for Bilingual Lexicon Extraction

    Directory of Open Access Journals (Sweden)

    Jae-Hoon Kim

    2015-01-01

    Full Text Available A pivot-based approach for bilingual lexicon extraction is based on the similarity of context vectors represented by words in a pivot language like English. In this paper, in order to show validity and usability of the pivot-based approach, we evaluate the approach in company with two different methods for estimating context vectors: one estimates them from two parallel corpora based on word association between source words (resp., target words and pivot words and the other estimates them from two parallel corpora based on word alignment tools for statistical machine translation. Empirical results on two language pairs (e.g., Korean-Spanish and Korean-French have shown that the pivot-based approach is very promising for resource-poor languages and this approach observes its validity and usability. Furthermore, for words with low frequency, our method is also well performed.

  17. Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops.

    Science.gov (United States)

    Christou, Anastasis; Georgiadou, Egli C; Filippou, Panagiota; Manganaris, George A; Fotopoulos, Vasileios

    2014-03-01

    Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops.

  18. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  19. Facial Feature Extraction Method Based on Coefficients of Variances

    Institute of Scientific and Technical Information of China (English)

    Feng-Xi Song; David Zhang; Cai-Kou Chen; Jing-Yu Yang

    2007-01-01

    Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) are two popular feature ex- traction techniques in statistical pattern recognition field. Due to small sample size problem LDA cannot be directly applied to appearance-based face recognition tasks. As a consequence, a lot of LDA-based facial feature extraction techniques are proposed to deal with the problem one after the other. Nullspace Method is one of the most effective methods among them. The Nullspace Method tries to find a set of discriminant vectors which maximize the between-class scatter in the null space of the within-class scatter matrix. The calculation of its discriminant vectors will involve performing singular value decomposition on a high-dimensional matrix. It is generally memory- and time-consuming. Borrowing the key idea in Nullspace method and the concept of coefficient of variance in statistical analysis we present a novel facial feature extraction method, i.e., Discriminant based on Coefficient of Variance (DCV) in this paper. Experimental results performed on the FERET and AR face image databases demonstrate that DCV is a promising technique in comparison with Eigenfaces, Nullspace Method, and other state-of-the-art facial feature extraction methods.

  20. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  1. RNACompress: Grammar-based compression and informational complexity measurement of RNA secondary structure

    Directory of Open Access Journals (Sweden)

    Chen Chun

    2008-03-01

    Full Text Available Abstract Background With the rapid emergence of RNA databases and newly identified non-coding RNAs, an efficient compression algorithm for RNA sequence and structural information is needed for the storage and analysis of such data. Although several algorithms for compressing DNA sequences have been proposed, none of them are suitable for the compression of RNA sequences with their secondary structures simultaneously. This kind of compression not only facilitates the maintenance of RNA data, but also supplies a novel way to measure the informational complexity of RNA structural data, raising the possibility of studying the relationship between the functional activities of RNA structures and their complexities, as well as various structural properties of RNA based on compression. Results RNACompress employs an efficient grammar-based model to compress RNA sequences and their secondary structures. The main goals of this algorithm are two fold: (1 present a robust and effective way for RNA structural data compression; (2 design a suitable model to represent RNA secondary structure as well as derive the informational complexity of the structural data based on compression. Our extensive tests have shown that RNACompress achieves a universally better compression ratio compared with other sequence-specific or common text-specific compression algorithms, such as Gencompress, winrar and gzip. Moreover, a test of the activities of distinct GTP-binding RNAs (aptamers compared with their structural complexity shows that our defined informational complexity can be used to describe how complexity varies with activity. These results lead to an objective means of comparing the functional properties of heteropolymers from the information perspective. Conclusion A universal algorithm for the compression of RNA secondary structure as well as the evaluation of its informational complexity is discussed in this paper. We have developed RNACompress, as a useful tool

  2. An RNA-Seq-based reference transcriptome for Citrus.

    Science.gov (United States)

    Terol, Javier; Tadeo, Francisco; Ventimilla, Daniel; Talon, Manuel

    2016-03-01

    Previous RNA-Seq studies in citrus have been focused on physiological processes relevant to fruit quality and productivity of the major species, especially sweet orange. Less attention has been paid to vegetative or reproductive tissues, while most Citrus species have never been analysed. In this work, we characterized the transcriptome of vegetative and reproductive tissues from 12 Citrus species from all main phylogenetic groups. Our aims were to acquire a complete view of the citrus transcriptome landscape, to improve previous functional annotations and to obtain genetic markers associated with genes of agronomic interest. 28 samples were used for RNA-Seq analysis, obtained from 12 Citrus species: C. medica, C. aurantifolia, C. limon, C. bergamia, C. clementina, C. deliciosa, C. reshni, C. maxima, C. paradisi, C. aurantium, C. sinensis and Poncirus trifoliata. Four different organs were analysed: root, phloem, leaf and flower. A total of 3421 million Illumina reads were produced and mapped against the reference C. clementina genome sequence. Transcript discovery pipeline revealed 3326 new genes, the number of genes with alternative splicing was increased to 19,739, and a total of 73,797 transcripts were identified. Differential expression studies between the four tissues showed that gene expression is overall related to the physiological function of the specific organs above any other variable. Variants discovery analysis revealed the presence of indels and SNPs in genes associated with fruit quality and productivity. Pivotal pathways in citrus such as those of flavonoids, flavonols, ethylene and auxin were also analysed in detail.

  3. An RNA-Seq-based reference transcriptome for Citrus.

    Science.gov (United States)

    Terol, Javier; Tadeo, Francisco; Ventimilla, Daniel; Talon, Manuel

    2016-03-01

    Previous RNA-Seq studies in citrus have been focused on physiological processes relevant to fruit quality and productivity of the major species, especially sweet orange. Less attention has been paid to vegetative or reproductive tissues, while most Citrus species have never been analysed. In this work, we characterized the transcriptome of vegetative and reproductive tissues from 12 Citrus species from all main phylogenetic groups. Our aims were to acquire a complete view of the citrus transcriptome landscape, to improve previous functional annotations and to obtain genetic markers associated with genes of agronomic interest. 28 samples were used for RNA-Seq analysis, obtained from 12 Citrus species: C. medica, C. aurantifolia, C. limon, C. bergamia, C. clementina, C. deliciosa, C. reshni, C. maxima, C. paradisi, C. aurantium, C. sinensis and Poncirus trifoliata. Four different organs were analysed: root, phloem, leaf and flower. A total of 3421 million Illumina reads were produced and mapped against the reference C. clementina genome sequence. Transcript discovery pipeline revealed 3326 new genes, the number of genes with alternative splicing was increased to 19,739, and a total of 73,797 transcripts were identified. Differential expression studies between the four tissues showed that gene expression is overall related to the physiological function of the specific organs above any other variable. Variants discovery analysis revealed the presence of indels and SNPs in genes associated with fruit quality and productivity. Pivotal pathways in citrus such as those of flavonoids, flavonols, ethylene and auxin were also analysed in detail. PMID:26261026

  4. Exploiting Nanotechnology for the Development of MicroRNA-Based Cancer Therapeutics.

    Science.gov (United States)

    Tyagi, Nikhil; Arora, Sumit; Deshmukh, Sachin K; Singh, Seema; Marimuthu, Saravanakumar; Singh, Ajay P

    2016-01-01

    MicroRNAs (miRNAs/miRs) represent a novel class of small non-coding RNAs that post-transcriptionally regulate gene expression by base pairing with complementary sequences in the 3' untranslated region (UTR) of target mRNAs. Functional studies suggest that miRNAs control almost every biological process, and their aberrant expression leads to a disease state, such as cancer. Differential expression of miRNAs in cancerous versus normal cells have generated enormous interest for the development of miRNA-based cancer cell-targeted therapeutics. Depending on the miRNA function and expression in cancer, two types of miRNA-based therapeutic strategies can be utilized that either restore or inhibit miRNA function through exogenous delivery of miRNAs mimics or inhibitors (anti-miRs). However, hydrophilic nature of miRNA mimics/anti-miRs, sensitivity to nuclease degradation in serum, poor penetration and reduced uptake by the tumor cells are chief hurdles in accomplishing their efficient in vivo delivery. To overcome these barriers, several nanotechnology-based systems are being developed and tested for delivery efficacy. This review summarizes the importance of miRNAs-based therapeutics in cancer, associated translational challenges and novel nanotechnology-assisted delivery systems that hold potential for next-generation miRNA-based cancer therapeutics. PMID:27301170

  5. Comparison of Two RNA Extraction Methods of Coconut Tree%2种提取椰子树RNA方法的比较研究

    Institute of Scientific and Technical Information of China (English)

    杨光华; 刘进平

    2011-01-01

    椰子树组织中富含多糖和酚类化合物,为了获取高质量的RNA,笔者以椰子树叶片和根部为材料,比较分析CTAB法和SDS法提取RNA的效果。结果表明:CTAB法提取椰子树组织的总RNA效果较好,能有效克服酚类物质和多糖对RNA提取的影响。经OD值检测和电泳检测,总RNA的28 S和18 S条带清晰可见,纯度较高,完整性好,无明显降解,以此RNA为模板进行RT-PCR反应,能获得特异条带,表明该RNA完全可以用于后续的分子生物学操作。%Tissue of coconut tree is rich in polysaccharides and phenolic compounds.In order to obtain high-quality RNA,comparison of SDS method and CTAB method for total RNA extraction from leaves and roots of coconut tree was conducted.The results showed that CTAB method was more suitable and effective than SDS method in the total RNA extraction of coconut trees and effectively overcame the problems caused by polyphenols and polysaccharides in the RNA extraction.As detected with OD value and gel electrophoresis,bands of 28S and 18S rRNA are clearly visible,with high purity,integrity and no degradation.With the RNAs as templates for RT-PCR reaction,specific bands were produced,indicating that the RNAs could be used for subsequent molecular biological operations.

  6. Quantitative detection of ING1 mRNA under different gene regulation based on molecular beacon

    Institute of Scientific and Technical Information of China (English)

    LIU Bin; WANG Kemin; XIAO Zhiqiang; WANG Wei; TAN Weihong; SUN Yi; TANG Hongxing; YANG Xiaohai

    2006-01-01

    The effects of drug treatment, gene transfection, and p53 RNA interference (RNAi) on level of ING1 mRNA in tumor cells were quantitatively detected with the help of ING1 molecular beacon (MB)/cRNA standard curve based on MB detection technology. Results showed that the level of ING1 mRNA could be upregulated by 5-FU treatment or ING1 transfection in low expression cell line of MCF-7 and be inhibited by silencing p53 with RNAi technology in normal expression cell line of CNE2. The level of ING1 varied from 7.7×10-16 to 53.4×10-16 mol/ug total RNA of tumor cells. The results not only provided evidence for the regulation effects of gene expression but also were applied to investigating the interaction of multigenes on signal transduction pathway.

  7. High-throughput-sequencing-based identification of a grapevine fanleaf virus satellite RNA in Vitis vinifera.

    Science.gov (United States)

    Chiumenti, Michela; Mohorianu, Irina; Roseti, Vincenzo; Saldarelli, Pasquale; Dalmay, Tamas; Minafra, Angelantonio

    2016-05-01

    A new satellite RNA (satRNA) of grapevine fanleaf virus (GFLV) was identified by high-throughput sequencing of high-definition (HD) adapter libraries from grapevine plants of the cultivar Panse precoce (PPE) affected by enation disease. The complete nucleotide sequence was obtained by automatic sequencing using primers designed based on next-generation sequencing (NGS) data. The full-length sequence, named satGFLV-PPE, consisted of 1119 nucleotides with a single open reading frame from position 15 to 1034. This satRNA showed maximum nucleotide sequence identity of 87 % to satArMV-86 and satGFLV-R6. Symptomatic grapevines were surveyed for the presence of the satRNA, and no correlation was found between detection of the satRNA and enation symptom expression. PMID:26873812

  8. Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls.

    Science.gov (United States)

    Foss, R D; Guha-Thakurta, N; Conran, R M; Gutman, P

    1994-09-01

    A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues. PMID:7981889

  9. Knowledge-based analysis of functional impacts of mutations in microRNA seed regions

    Indian Academy of Sciences (India)

    Anindya Bhattacharya; Yan Cui

    2015-10-01

    MicroRNAs are a class of important post-transcriptional regulators. Genetic and somatic mutations in miRNAs, especially those in the seed regions, have profound and broad impacts on gene expression and physiological and pathological processes. Over 500 SNPs were mapped to the miRNA seeds, which are located at position 2–8 of the mature miRNA sequences. We found that the central positions of the miRNA seeds contain fewer genetic variants and therefore are more evolutionary conserved than the peripheral positions in the seeds. We developed a knowledge-based method to analyse the functional impacts of mutations in miRNA seed regions. We computed the gene ontology-based similarity score GOSS and the GOSS percentile score for all 517 SNPs in miRNA seeds. In addition to the annotation of SNPs for their functional effects, in the present article we also present a detailed analysis pipeline for finding the key functional changes for seed SNPs. We performed a detailed gene ontology graph-based analysis of enriched functional categories for miRNA target gene sets. In the analysis of a SNP in the seed region of hsa-miR-96 we found that two key biological processes for progressive hearing loss `Neurotrophin TRK receptor signaling pathway' and `Epidermal growth factor receptor signaling pathway' were significantly and differentially enriched by the two sets of allele-specific target genes of miRNA hsa-miR-96.

  10. Does base-pairing strength play a role in microRNA repression?

    Science.gov (United States)

    Carmel, Ido; Shomron, Noam; Heifetz, Yael

    2012-11-01

    MicroRNAs (miRNAs) are short, single-stranded RNAs that silence gene expression by either degrading mRNA or repressing translation. Each miRNA regulates a specific set of mRNA "targets" by binding to complementary sequences in their 3' untranslated region. In this study, we examined the importance of the base-pairing strength of the miRNA-target duplex to repression. We hypothesized that if base-pairing strength affects the functionality of miRNA repression, organisms with higher body temperature or that live at higher temperatures will have miRNAs with higher G/C content so that the miRNA-target complex will remain stable. In the nine model organisms examined, we found a significant correlation between the average G/C content of miRNAs and physiological temperature, supporting our hypothesis. Next, for each organism examined, we compared the average G/C content of miRNAs that are conserved among distant organisms and that of miRNAs that are evolutionarily recent. We found that the average G/C content of ancient miRNAs is lower than recent miRNAs in homeotherms, whereas the trend was inversed in poikilotherms, suggesting that G/C content is associated with temperature, thus further supporting our hypothesis. In the organisms examined, the average G/C content of miRNA "seed" sequences was higher than that of mature miRNAs, which was higher than pre-miRNA loops, suggesting an association between the degree of functionality of the sequence and its average G/C content. Our analyses show a possible association between the base-pairing strength of miRNA-targets and the temperature of an organism, suggesting that base-pairing strength plays a role in repression by miRNAs. PMID:23019592

  11. Nanotechnology-based strategies for the detection and quantification of microRNA.

    Science.gov (United States)

    Degliangeli, Federica; Pompa, Pier Paolo; Fiammengo, Roberto

    2014-07-28

    MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.

  12. Base-pairing versatility determines wobble sites in tRNA anticodons of vertebrate mitogenomes.

    Directory of Open Access Journals (Sweden)

    Miguel M Fonseca

    Full Text Available BACKGROUND: Vertebrate mitochondrial genomes typically have one transfer RNA (tRNA for each synonymous codon family. This limited anticodon repertoire implies that each tRNA anticodon needs to wobble (establish a non-Watson-Crick base pairing between two nucleotides in RNA molecules to recognize one or more synonymous codons. Different hypotheses have been proposed to explain the factors that determine the nucleotide composition of wobble sites in vertebrate mitochondrial tRNA anticodons. Until now, the two major postulates--the "codon-anticodon adaptation hypothesis" and the "wobble versatility hypothesis"--have not been formally tested in vertebrate mitochondria because both make the same predictions regarding the composition of anticodon wobble sites. The same is true for the more recent "wobble cost hypothesis". PRINCIPAL FINDINGS: In this study we have analyzed the occurrence of synonymous codons and tRNA anticodon wobble sites in 1553 complete vertebrate mitochondrial genomes, focusing on three fish species with mtDNA codon usage bias reversal (L-strand is GT-rich. These mitogenomes constitute an excellent opportunity to study the evolution of the wobble nucleotide composition of tRNA anticodons because due to the reversal the predictions for the anticodon wobble sites differ between the existing hypotheses. We observed that none of the wobble sites of tRNA anticodons in these unusual mitochondrial genomes coevolved to match the new overall codon usage bias, suggesting that nucleotides at the wobble sites of tRNA anticodons in vertebrate mitochondrial genomes are determined by wobble versatility. CONCLUSIONS/SIGNIFICANCE: Our results suggest that, at wobble sites of tRNA anticodons in vertebrate mitogenomes, selection favors the most versatile nucleotide in terms of wobble base-pairing stability and that wobble site composition is not influenced by codon usage. These results are in agreement with the "wobble versatility hypothesis".

  13. 番茄叶片小RNA提取方法的优化%Majorization of small RNA extraction methods in tomato leaf

    Institute of Scientific and Technical Information of China (English)

    陈华; 昌伟; 杨瑞; 王绍辉

    2012-01-01

    This experiment used tomato leaf to extract small RNA.UNIQ-10 Column Trizol Extraction Kit+LiCl method,a general Trizol+ LiClmethod and the improved Trizol + LiCl method were compared on the extraction effect.The results showed that the improved Trizol+LiCl method could extract high-quality small RNA from tomato leaves,Agarose gel electro-phoresis of total RNA showed clear and complete strips,nucleic acid protein detector measured the 1 026.8 ng/μL in concentration,2.00 for A260/A280 and 2.31 for A260/A230.15% polyacrylamide - 7 M urea gel electrophoresis showed clear small RNA strips area.The high-quality small RNA from tomato leaves by improved Trizol+LiCl method met the criterion for small RNA Northern blot and other molecular biology experiments.%以番茄叶片为材料提取小RNA,比较分析UNlQ-10柱式Trizol总RNA抽提试剂盒+LiC1法,常规Tr-izol+LiC1法和改进的Trizol法+LiC1法的提取效果.结果表明,改进的Trizol+ LiCl法能从番茄叶片中提取高质量的小RNA,总RNA琼脂糖凝胶电泳条带完整清晰,核酸蛋白测定仪显示RNA的浓度为1 026.8 ng/μL,A260/A280比值为2.00;A260/A230比值为2.31.15%聚丙烯酰胺—7 mol/L尿素凝胶电泳显示小RNA带型区域清晰.用该方法提取的高质量的番茄叶片小RNA符合RT-PCR,Northern blot等分子生物学试验的标准.

  14. SSVEP Extraction Based on the Similarity of Background EEG

    OpenAIRE

    Zhenghua Wu

    2014-01-01

    Steady-state Visual Evoked Potential (SSVEP) outperforms the other types of ERPs for Brain-computer Interface (BCI), and thus it is widely employed. In order to apply SSVEP-based BCI to real life situations, it is important to improve the accuracy and transfer rate of the system. Aimed at this target, many SSVEP extraction methods have been proposed. All these methods are based directly on the properties of SSVEP, such as power and phase. In this study, we first filtered out the target freque...

  15. Key frame extraction based on spatiotemporal motion trajectory

    Science.gov (United States)

    Zhang, Yunzuo; Tao, Ran; Zhang, Feng

    2015-05-01

    Spatiotemporal motion trajectory can accurately reflect the changes of motion state. Motivated by this observation, this letter proposes a method for key frame extraction based on motion trajectory on the spatiotemporal slice. Different from the well-known motion related methods, the proposed method utilizes the inflexions of the motion trajectory on the spatiotemporal slice of all the moving objects. Experimental results show that although a similar performance is achieved in the single-objective screen, by comparing the proposed method to that achieved with the state-of-the-art methods based on motion energy or acceleration, the proposed method shows a better performance in a multiobjective video.

  16. The ReNAissanCe of mRNA-based cancer therapy.

    Science.gov (United States)

    Van Lint, Sandra; Renmans, Dries; Broos, Katrijn; Dewitte, Heleen; Lentacker, Ine; Heirman, Carlo; Breckpot, Karine; Thielemans, Kris

    2015-02-01

    About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity. The interest in direct application of mRNA experienced a revival, as researchers became aware of the many advantages mRNA offers. Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. In this review, we highlight this renaissance using a timeline providing all milestones in the application of mRNA for anticancer vaccination.

  17. Term Recognition and Extraction based on Semantics for Ontology Construction

    Directory of Open Access Journals (Sweden)

    Akalya.B

    2012-03-01

    Full Text Available In recent years the development of Ontologys, leads to explicit formal specifications of the terms in the domain and relations among them. The construction of Ontology often requires a domain specific corpus in conceptualizing the domain knowledge. It is an indispensible task to identify a list of significant terms for constructing a Structured Ontology. In this paper, we investigate the use of Semantic Similarity-based metrics for term recognition and extraction, for ontology construction from the text document. The methodology uses Taxonomy and Wikipedia to reinforce the automatic term recognition and extraction from structured documents. It is done with the assumption of candidate terms for a topic are often associated with its topic-specific keywords through Semantic Similarity-based metrics by making use of WordNet. A hierarchical relationship of super-topics and sub-topics is defined by Taxonomy, meanwhile, Wikipedia is used to provide a semantic relationship and background knowledge for topics that are defined in the Taxonomy to surpervise the term recognition and extraction. Experimental results show that the proposed methodology is viable to be applied in a small corpus supporting Ontology construction, which renders the foundation for higher recall and precision, when compared with existing methodologies.

  18. Motion feature extraction scheme for content-based video retrieval

    Science.gov (United States)

    Wu, Chuan; He, Yuwen; Zhao, Li; Zhong, Yuzhuo

    2001-12-01

    This paper proposes the extraction scheme of global motion and object trajectory in a video shot for content-based video retrieval. Motion is the key feature representing temporal information of videos. And it is more objective and consistent compared to other features such as color, texture, etc. Efficient motion feature extraction is an important step for content-based video retrieval. Some approaches have been taken to extract camera motion and motion activity in video sequences. When dealing with the problem of object tracking, algorithms are always proposed on the basis of known object region in the frames. In this paper, a whole picture of the motion information in the video shot has been achieved through analyzing motion of background and foreground respectively and automatically. 6-parameter affine model is utilized as the motion model of background motion, and a fast and robust global motion estimation algorithm is developed to estimate the parameters of the motion model. The object region is obtained by means of global motion compensation between two consecutive frames. Then the center of object region is calculated and tracked to get the object motion trajectory in the video sequence. Global motion and object trajectory are described with MPEG-7 parametric motion and motion trajectory descriptors and valid similar measures are defined for the two descriptors. Experimental results indicate that our proposed scheme is reliable and efficient.

  19. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.

    Science.gov (United States)

    Lacerenza, D; Aneli, S; Omedei, M; Gino, S; Pasino, S; Berchialla, P; Robino, C

    2016-05-01

    "Touch DNA" refers to the DNA that is left behind when a person touches or comes into contact with an item. However, the source of touch DNA is still debated and the large variability in DNA yield from casework samples suggests that, besides skin, various body fluids can be transferred through contact. Another important issue concerning touch DNA is the possible occurrence of secondary transfer, but the data published in the literature in relation to the background levels of foreign DNA present on the hand surfaces of the general population are very limited. As the present study aimed at better understanding the nature and characteristics of touch DNA, samples were collected from the palmar surface of the hands and fingers ("PHF" samples) of 30 male and 30 female donors by tape-lifting/swabbing and subjected to DNA/RNA co-extraction. Multiplex mRNA profiling showed that cellular material different from skin could be observed in 15% of the PHF samples. The total amount of DNA recovered from these samples (median 5.1 ng) was significantly higher than that obtained from samples containing skin cells only (median 1.6 ng). The integrity of the DNA isolated from the donors' hands and fingers as well as the prevalence of DNA mixtures were evaluated by STR typing and compared with reference STR profiles from buccal swabs. DNA integrity appeared significantly higher in the male rather than in the female subsample, as the average percentage of the donors' alleles effectively detected in PHF profiles was 75.1% and 60.1%, respectively. The prevalence of mixtures with a foreign DNA contribution ≥20% was 19.2% (30.0% in the female PHF samples and 8.3% in the male PHF samples). The obtained results support the hypothesis that transfer of cellular material different from skin may underlie the occasional recovery of quality STR profiles from handled items. These results also suggest that gender may represent an important factor influencing the propensity of individuals to carry

  20. Features Extraction for Object Detection Based on Interest Point

    Directory of Open Access Journals (Sweden)

    Amin Mohamed Ahsan

    2013-05-01

    Full Text Available In computer vision, object detection is an essential process for further processes such as object tracking, analyzing and so on. In the same context, extraction features play important role to detect the object correctly. In this paper we present a method to extract local features based on interest point which is used to detect key-points within an image, then, compute histogram of gradient (HOG for the region surround that point. Proposed method used speed-up robust feature (SURF method as interest point detector and exclude the descriptor. The new descriptor is computed by using HOG method. The proposed method got advantages of both mentioned methods. To evaluate the proposed method, we used well-known dataset which is Caltech101. The initial result is encouraging in spite of using a small data for training.

  1. Modification of evidence theory based on feature extraction

    Institute of Scientific and Technical Information of China (English)

    DU Feng; SHI Wen-kang; DENG Yong

    2005-01-01

    Although evidence theory has been widely used in information fusion due to its effectiveness of uncertainty reasoning, the classical DS evidence theory involves counter-intuitive behaviors when high conflict information exists. Many modification methods have been developed which can be classified into the following two kinds of ideas, either modifying the combination rules or modifying the evidence sources. In order to make the modification more reasonable and more effective, this paper gives a thorough analysis of some typical existing modification methods firstly, and then extracts the intrinsic feature of the evidence sources by using evidence distance theory. Based on the extracted features, two modified plans of evidence theory according to the corresponding modification ideas have been proposed. The results of numerical examples prove the good performance of the plans when combining evidence sources with high conflict information.

  2. Music snippet extraction via melody-based repeated pattern discovery

    Institute of Scientific and Technical Information of China (English)

    XU JiePing; ZHAO Yang; CHEN Zhe; LIU ZiLi

    2009-01-01

    In this paper, we present a complete set of procedures to automatically extract a music snippet, defined as the most representative or the highlighted excerpt of a music clip. We first generate a modified and compact similarity matrix based on selected features and distance metrics, and then several improved techniques for music repeated pattern discovery are utilized because a music snippet is usually a part of the repeated melody, main theme or chorus. During the process, redundant and wrongly detected patterns are discarded, boundaries are corrected using beat information, and final clusters are also further sorted according to the occurrence frequency and energy information. Subsequently, following our methods, we designed a music snippet extraction system which allows users to detect snippets. Experiments performed on the system show the superiority of our proposed approach.

  3. Spindle extraction method for ISAR image based on Radon transform

    Science.gov (United States)

    Wei, Xia; Zheng, Sheng; Zeng, Xiangyun; Zhu, Daoyuan; Xu, Gaogui

    2015-12-01

    In this paper, a method of spindle extraction of target in inverse synthetic aperture radar (ISAR) image is proposed which depends on Radon Transform. Firstly, utilizing Radon Transform to detect all straight lines which are collinear with these line segments in image. Then, using Sobel operator to detect image contour. Finally, finding all intersections of each straight line and image contour, the two intersections which have maximum distance between them is the two ends of this line segment and the longest line segment of all line segments is spindle of target. According to the proposed spindle extraction method, one hundred simulated ISAR images which are respectively rotated 0 degrees, 10 degrees, 20 degrees, 30 degrees and 40 degrees in counterclockwise are used to do experiment and the proposed method and the detection results are more close to the real spindle of target than the method based on Hough Transform .

  4. Remote Sensing Image Feature Extracting Based Multiple Ant Colonies Cooperation

    Directory of Open Access Journals (Sweden)

    Zhang Zhi-long

    2014-02-01

    Full Text Available This paper presents a novel feature extraction method for remote sensing imagery based on the cooperation of multiple ant colonies. First, multiresolution expression of the input remote sensing imagery is created, and two different ant colonies are spread on different resolution images. The ant colony in the low-resolution image uses phase congruency as the inspiration information, whereas that in the high-resolution image uses gradient magnitude. The two ant colonies cooperate to detect features in the image by sharing the same pheromone matrix. Finally, the image features are extracted on the basis of the pheromone matrix threshold. Because a substantial amount of information in the input image is used as inspiration information of the ant colonies, the proposed method shows higher intelligence and acquires more complete and meaningful image features than those of other simple edge detectors.

  5. Antioxidant Activity and Induction of mRNA Expressions of Antioxidant Enzymes in HEK-293 Cells of Moringa oleifera Leaf Extract.

    Science.gov (United States)

    Vongsak, Boonyadist; Mangmool, Supachoke; Gritsanapan, Wandee

    2015-08-01

    The leaves of Moringa oleifera, collected in different provinces in Thailand, were determined for the contents of total phenolics, total flavonoids, major components, and antioxidant activity. The extract and its major active components were investigated for the inhibition of H2O2-induced reactive oxygen species production and the effects on antioxidant enzymes mRNA expression. The extract, crypto-chlorogenic acid, isoquercetin and astragalin, significantly reduced the reactive oxygen species production inducing by H2O2 in HEK-293 cells. Treatment with isoquercetin significantly increased the mRNA expression levels of antioxidant enzymes such as superoxide dismutase, catalase and heme oxygenase 1. These results confirm that M. oleifera leaves are good sources of natural antioxidant with isoquercetin as an active compound. PMID:26166137

  6. Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction.

    Science.gov (United States)

    Koid, Amy; Nelson, William C; Mraz, Amy; Heidelberg, Karla B

    2012-06-01

    Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

  7. Antioxidant Activity and Induction of mRNA Expressions of Antioxidant Enzymes in HEK-293 Cells of Moringa oleifera Leaf Extract.

    Science.gov (United States)

    Vongsak, Boonyadist; Mangmool, Supachoke; Gritsanapan, Wandee

    2015-08-01

    The leaves of Moringa oleifera, collected in different provinces in Thailand, were determined for the contents of total phenolics, total flavonoids, major components, and antioxidant activity. The extract and its major active components were investigated for the inhibition of H2O2-induced reactive oxygen species production and the effects on antioxidant enzymes mRNA expression. The extract, crypto-chlorogenic acid, isoquercetin and astragalin, significantly reduced the reactive oxygen species production inducing by H2O2 in HEK-293 cells. Treatment with isoquercetin significantly increased the mRNA expression levels of antioxidant enzymes such as superoxide dismutase, catalase and heme oxygenase 1. These results confirm that M. oleifera leaves are good sources of natural antioxidant with isoquercetin as an active compound.

  8. Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

    OpenAIRE

    Oliveira, A. C. N.; Raemdonck, Koen; Martens, Thomas; Rombouts, Koen; Simón-Vázquez, Rosana; Botelho, C. M.; Lopes, Ivo Edgar Araújo; Lúcio, M.; González-Fernández, África; Real Oliveira, M. Elisabete C.D.; Gomes, Andreia; Braeckmans, Kevin

    2015-01-01

    While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabili...

  9. Identification of dengue viral RNA-dependent RNA polymerase inhibitor using computational fragment-based approaches and molecular dynamics study.

    Science.gov (United States)

    Anusuya, Shanmugam; Velmurugan, Devadasan; Gromiha, M Michael

    2016-07-01

    Dengue is a major public health concern in tropical and subtropical countries of the world. There are no specific drugs available to treat dengue. Even though several candidates targeted both viral and host proteins to overcome dengue infection, they have not yet entered into the later stages of clinical trials. In order to design a drug for dengue fever, newly emerged fragment-based drug designing technique was applied. RNA-dependent RNA polymerase, which is essential for dengue viral replication is chosen as a drug target for dengue drug discovery. A cascade of methods, fragment screening, fragment growing, and fragment linking revealed the compound [2-(4-carbamoylpiperidin-1-yl)-2-oxoethyl]8-(1,3-benzothiazol-2-yl)naphthalene-1-carboxylate as a potent dengue viral polymerase inhibitor. Both strain energy and binding free energy calculations predicted that this could be a better inhibitor than the existing ones. Molecular dynamics simulation studies showed that the dengue polymerase-lead complex is stable and their interactions are consistent throughout the simulation. The hydrogen-bonded interactions formed by the residues Arg792, Thr794, Ser796, and Asn405 are the primary contributors for the stability and the rigidity of the polymerase-lead complex. This might keep the polymerase in closed conformation and thus inhibits viral replication. Hence, this might be a promising lead molecule for dengue drug designing. Further optimization of this lead molecule would result in a potent drug for dengue. PMID:26262439

  10. 油茶总RNA的提取与内参基因的初选%RNA extraction and primary screening of reference genes in Camellia oleifera

    Institute of Scientific and Technical Information of China (English)

    宋志波; 刘敏; 贾宝光; 田雷; 曾艳玲; 周俊琴; 谭晓风; 张琳

    2014-01-01

    为得到高质量的油茶总RNA及稳定的内参基因,以油茶良种‘华硕’为试验材料,采用ambion试剂盒结合CTAB法进行总RNA提取。获得的总RNA完整性好,纯度高;从前期构建的油茶转录组数据中选取ACT(肌动蛋白基因)等14个基因作为候选内参基因,共设计22对引物,通过RT-PCR反应体系的优化及琼脂糖凝胶电泳检测,快速筛选出了适合油茶不同组织(器官)的内参基因。ALB(清蛋白基因),EF1α(延伸因子基因),ETIF3H(启动因子基因),UBC2(泛素结合酶基因)可以作为油茶果实初选内参基因,EF1α可以作为油茶根、茎、花及果实内参基因,ETIF3H可以作为油茶根、茎、叶、花瓣的内参基因。%In order to obtain high quality total RNA and stable reference genes in Camellia oleifera, taking ifne cultivar‘Huashuo’ as materials, total RNA was extracted by a combination of ambion kit and CTAB method. The obtained total RNA has good integrity and high purity. A total of 14 genes were selected as candidates of reference genes based on the previously constructed transcriptome data in C. oleifera, and 22 primers were designed for the 14 genes. The reference genes suitable for different tissues or organs were rapidly screened out by RT-PCR reaction system optimization and agarose gel electrophoresis detection. ALB (albumin gene), EF1α (elongation factor gene), ETIF3H (eukaryotic translation initiation gene), UBC2 (ubiquitin-conjugating enzyme gene) can be used as the reference genes for fruits, EF1αcan be used as a reference gene for root, stem, lfower and fruit, and ETIF3H can be used as a reliable reference gene for root, stem, leaf and fruit in C. oleifera.

  11. Analogy between gambling and measurement-based work extraction

    Science.gov (United States)

    Vinkler, Dror A.; Permuter, Haim H.; Merhav, Neri

    2016-04-01

    In information theory, one area of interest is gambling, where mutual information characterizes the maximal gain in wealth growth rate due to knowledge of side information; the betting strategy that achieves this maximum is named the Kelly strategy. In the field of physics, it was recently shown that mutual information can characterize the maximal amount of work that can be extracted from a single heat bath using measurement-based control protocols, i.e. using ‘information engines’. However, to the best of our knowledge, no relation between gambling and information engines has been presented before. In this paper, we briefly review the two concepts and then demonstrate an analogy between gambling, where bits are converted into wealth, and information engines, where bits representing measurements are converted into energy. From this analogy follows an extension of gambling to the continuous-valued case, which is shown to be useful for investments in currency exchange rates or in the stock market using options. Moreover, the analogy enables us to use well-known methods and results from one field to solve problems in the other. We present three such cases: maximum work extraction when the probability distributions governing the system and measurements are unknown, work extraction when some energy is lost in each cycle, e.g. due to friction, and an analysis of systems with memory. In all three cases, the analogy enables us to use known results in order to obtain new ones.

  12. Leaf Vein Extraction Based on Gray-scale Morphology

    Directory of Open Access Journals (Sweden)

    Xiaodong Zheng

    2010-12-01

    Full Text Available Leaf features play an important role in plant species identification and plant taxonomy. The type of the leaf vein is an important morphological feature of the leaf in botany. Leaf vein should be extracted from the leaf in the image before discriminating its type. In this paper a new method of leaf vein extraction has been proposed based on gray-scale morphology. Firstly, the color image of the plant leaf is transformed to the gray image according to the hue and intensity information. Secondly, the gray-scale morphology processing is applied to the image to eliminate the color overlap in the whole leaf vein and the whole background. Thirdly, the linear intensity adjustment is adopted to enlarge the gray value difference between the leaf vein and its background. Fourthly, calculate a threshold with OSTU method to segment the leaf vein from its background. Finally, the leaf vein can be got after some processing on details. Experiments have been conducted with several images. The results show the effectiveness of the method. The idea of the method is also applicable to other linear objects extraction.

  13. Mechanism of gold solvent extraction from aurocyanide solution by quaternary amines: models of extracting species based on hydrogen bonding

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mechanism of gold solvent extraction from KAu(CN)2 solution was investigated by means of FTIR, EXAFS, ICP and radioactive tracer methods. Two extraction systems were studied, namely N263-tributyl phosphate(TBP)-n-dodecane and N263-iso-octanol-n-dodecane. High-reso- lution FT IR spectroscopy indicated that the CN stretching vibrations of the two extraction systems differred greatly. In order to interpret the significant difference in CN stretching vibrations, two extracting species models are proposed supramolecular structures based on the formation of hydrogen bonds between Au(CN)2- and modifiers such as TBP and iso-octanol.

  14. Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    OpenAIRE

    Adam Kotorashvili; Andrew Ramnauth; Christina Liu; Juan Lin; Kenny Ye; Ryung Kim; Rachel Hazan; Thomas Rohan; Susan Fineberg; Olivier Loudig

    2012-01-01

    BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or l...

  15. Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds

    OpenAIRE

    Rajakani, Raja; Narnoliya, Lokesh; Sangwan, Neelam Singh; Sangwan, Rajender Singh; Gupta, Vikrant

    2013-01-01

    Background High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it faile...

  16. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

    Science.gov (United States)

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

    2012-07-11

    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  17. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

    Science.gov (United States)

    Castiglione, Francesca; Rossi Degl'Innocenti, Duccio; Taddei, Antonio; Garbini, Francesca; Buccoliero, Anna Maria; Raspollini, Maria Rosaria; Pepi, Monica; Paglierani, Milena; Asirelli, Grazia; Freschi, Giancarlo; Bechi, Paolo; Taddei, Gian Luigi

    2007-09-01

    In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

  18. MAGIA, a web-based tool for miRNA and Genes Integrated Analysis.

    Science.gov (United States)

    Sales, Gabriele; Coppe, Alessandro; Bisognin, Andrea; Biasiolo, Marta; Bortoluzzi, Stefania; Romualdi, Chiara

    2010-07-01

    MAGIA (miRNA and genes integrated analysis) is a novel web tool for the integrative analysis of target predictions, miRNA and gene expression data. MAGIA is divided into two parts: the query section allows the user to retrieve and browse updated miRNA target predictions computed with a number of different algorithms (PITA, miRanda and Target Scan) and Boolean combinations thereof. The analysis section comprises a multistep procedure for (i) direct integration through different functional measures (parametric and non-parametric correlation indexes, a variational Bayesian model, mutual information and a meta-analysis approach based on P-value combination) of mRNA and miRNA expression data, (ii) construction of bipartite regulatory network of the best miRNA and mRNA putative interactions and (iii) retrieval of information available in several public databases of genes, miRNAs and diseases and via scientific literature text-mining. MAGIA is freely available for Academic users at http://gencomp.bio.unipd.it/magia.

  19. A Meta-Path-Based Prediction Method for Human miRNA-Target Association

    Science.gov (United States)

    Huang, Cong; Ding, Pingjian

    2016-01-01

    MicroRNAs (miRNAs) are short noncoding RNAs that play important roles in regulating gene expressing, and the perturbed miRNAs are often associated with development and tumorigenesis as they have effects on their target mRNA. Predicting potential miRNA-target associations from multiple types of genomic data is a considerable problem in the bioinformatics research. However, most of the existing methods did not fully use the experimentally validated miRNA-mRNA interactions. Here, we developed RMLM and RMLMSe to predict the relationship between miRNAs and their targets. RMLM and RMLMSe are global approaches as they can reconstruct the missing associations for all the miRNA-target simultaneously and RMLMSe demonstrates that the integration of sequence information can improve the performance of RMLM. In RMLM, we use RM measure to evaluate different relatedness between miRNA and its target based on different meta-paths; logistic regression and MLE method are employed to estimate the weight of different meta-paths. In RMLMSe, sequence information is utilized to improve the performance of RMLM. Here, we carry on fivefold cross validation and pathway enrichment analysis to prove the performance of our methods. The fivefold experiments show that our methods have higher AUC scores compared with other methods and the integration of sequence information can improve the performance of miRNA-target association prediction.

  20. Probabilistic contour extraction based on shape prior model

    Institute of Scientific and Technical Information of China (English)

    FAN Xin; LIANG De-qun

    2005-01-01

    Statistical shape prior model is employed to construct the dynamics in probabilistic contour estimation.By applying principal component analysis,plausible shape samples are efficiently generated to predict contour samples.Based on the shape-dependent dynamics and probabilistic image model,a particle filter is used to estimate the contour with a specific shape.Compared with the deterministic approach with shape information,the proposed method is simple yet more effective in extracting contours from images with shape variations and occlusion.

  1. Kernel-Based Learning for Domain-Specific Relation Extraction

    Science.gov (United States)

    Basili, Roberto; Giannone, Cristina; Del Vescovo, Chiara; Moschitti, Alessandro; Naggar, Paolo

    In a specific process of business intelligence, i.e. investigation on organized crime, empirical language processing technologies can play a crucial role. The analysis of transcriptions on investigative activities, such as police interrogatories, for the recognition and storage of complex relations among people and locations is a very difficult and time consuming task, ultimately based on pools of experts. We discuss here an inductive relation extraction platform that opens the way to much cheaper and consistent workflows. The presented empirical investigation shows that accurate results, comparable to the expert teams, can be achieved, and parametrization allows to fine tune the system behavior for fitting domain-specific requirements.

  2. Phase shift extraction algorithm based on Euclidean matrix norm.

    Science.gov (United States)

    Deng, Jian; Wang, Hankun; Zhang, Desi; Zhong, Liyun; Fan, Jinping; Lu, Xiaoxu

    2013-05-01

    In this Letter, the character of Euclidean matrix norm (EMN) of the intensity difference between phase-shifting interferograms, which changes in sinusoidal form with the phase shifts, is presented. Based on this character, an EMN phase shift extraction algorithm is proposed. Both the simulation calculation and experimental research show that the phase shifts with high precision can be determined with the proposed EMN algorithm easily. Importantly, the proposed EMN algorithm will supply a powerful tool for the rapid calibration of the phase shifts.

  3. VISUAL ATTENTION BASED KEYFRAMES EXTRACTION AND VIDEO SUMMARIZATION

    Directory of Open Access Journals (Sweden)

    P.Geetha

    2012-05-01

    Full Text Available Recent developments in digital video and drastic increase of internet use have increased the amount of people searching and watching videos online. In order to make the search of the videos easy, Summary of the video may be provided along with each video. The video summary provided thus should be effective so that the user would come to know the content of the video without having to watch it fully. The summary produced should consists of the key frames that effectively express the content and context of the video. This work suggests a method to extract key frames which express most of the information in the video. This is achieved by quantifying Visual attention each frame commands. Visual attention of each frame is quantified using a descriptor called Attention quantifier. This quantification of visual attention is based on the human attention mechanism that indicates color conspicuousness and the motion involved seek more attention. So based on the color conspicuousness and the motion involved each frame is given a Attention parameter. Based on the attention quantifier value the key frames are extracted and are summarized adaptively. This framework suggests a method to produces meaningful video summary.

  4. Study on Total RNA Extraction Methods from Miracle Fruit%神秘果果实总RNA提取方法比较研究

    Institute of Scientific and Technical Information of China (English)

    谭才邓; 李静; 邓毛程

    2013-01-01

      以神秘果果实为材料,以获得高质量RNA为目的,以针对性地去除多糖、酚类物质为出发点,比较了Trizol法、CTAB法,并相应加以改良。结果显示:CTAB粗提法方便快捷,CTAB精提法提取的总RNA完整性好、纯度高,RT-PCR反应表明提取的总RNA都能满足分子生物学实验的要求。%  In order to extract total RNA form miracle fruit and reduce the influence of polysaccharides and polyphenolics, we studied and compared four RNA extraction methods such as Trizol method, Trizol improved method, CTAB crude method and CTAB refined method. The results showed that: The CTAB crude extraction method was convenient and quick, total RAN from the CTAB refined method was proved to be relatively intact and high purity, RT-PCR analysis showed that the total RNA from both CTAB methods could be used in further molecular biology research.

  5. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    LENUS (Irish Health Repository)

    Mee, Blanaid C.

    2011-12-29

    The Saint James\\'s Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260\\/280 values using QIAamp were 33.2 ng\\/μL and 1.86, respectively, and using AllPrep were 23.2 ng\\/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng\\/μL and 8.16, respectively, and with AllPrep were 74.8 ng\\/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

  6. An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp.tritici

    Institute of Scientific and Technical Information of China (English)

    MA Li-Jie; QIAO Jia-xing; KONG Xin-yu; WANG Jun-juan; XU Xiang-ming; HU Xiao-ping

    2016-01-01

    Stripe rust, caused byPuccinia striformis f. sp.tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation ofP. striformisf. sp. triticiwinter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR (qRT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefifcient. Thus, we modiifed three previously reported RNA extraction methods with common and low-cost reagents (LiCl, SDS and NaCl) to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCl method was proven to be the best for extracting RNA from urediniospores of and wheat leaves infected byP. striformisf. sp. tritici, although the modiifed LiCl and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCl method has the folowing advantages: 1) Complete transfer of urediniospores ofP. striformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2) the use of low-cost NaCl to replace more expensive Trizol can reduce the cost; 3) the yield and quality of RNA can be increased;4) the improved method is more suitable for a large number and high quantity of samples from ifelds. Using the improved NaCl method, the amount of RNA was increased three times from urediniospores ofP. striformis f. sp. triticicompared from the extraction kit. Approximately, 10.11 μg total

  7. Knowledge-based reasoning to annotate noncoding RNA using multi-agent system.

    Science.gov (United States)

    Arruda, Wosley C; Souza, Daniel S; Ralha, Célia G; Walter, Maria Emilia M T; Raiol, Tainá; Brigido, Marcelo M; Stadler, Peter F

    2015-12-01

    Noncoding RNAs (ncRNAs) have been focus of intense research over the last few years. Since characteristics and signals of ncRNAs are not entirely known, researchers use different computational tools together with their biological knowledge to predict putative ncRNAs. In this context, this work presents ncRNA-Agents, a multi-agent system to annotate ncRNAs based on the output of different tools, using inference rules to simulate biologists' reasoning. Experiments with data from the fungus Saccharomyces cerevisiae allowed to measure the performance of ncRNA-Agents, with better sensibility, when compared to Infernal, a widely used tool for annotating ncRNA. Besides, data of the Schizosaccharomyces pombe and Paracoccidioides brasiliensis fungi identified novel putative ncRNAs, which demonstrated the usefulness of our approach. NcRNA-Agents can be be found at: http://www.biomol.unb.br/ncrna-agents.

  8. Reproducible analysis of sequencing-based RNA structure probing data with user-friendly tools

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan; Sidiropoulos, Nikos; Vinther, Jeppe

    2015-01-01

    time also made analysis of the data challenging for scientists without formal training in computational biology. Here, we discuss different strategies for data analysis of massive parallel sequencing-based structure-probing data. To facilitate reproducible and standardized analysis of this type of data......RNA structure-probing data can improve the prediction of RNA secondary and tertiary structure and allow structural changes to be identified and investigated. In recent years, massive parallel sequencing has dramatically improved the throughput of RNA structure probing experiments, but at the same......, we have made a collection of tools, which allow raw sequencing reads to be converted to normalized probing values using different published strategies. In addition, we also provide tools for visualization of the probing data in the UCSC Genome Browser and for converting RNA coordinates to genomic...

  9. RNA structure-based ribosome recruitment: lessons from the Dicistroviridae intergenic region IRESes.

    Science.gov (United States)

    Pfingsten, Jennifer S; Kieft, Jeffrey S

    2008-07-01

    In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5' end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by many viruses of medical and economic importance. In this review, we briefly describe and compare mechanistic and structural groups of viral IRES RNAs, focusing on those IRESes that are capable of direct ribosome recruitment using specific RNA structures. We then discuss in greater detail some recent advances in our understanding of the intergenic region IRESes of the Dicistroviridae, which use the most streamlined ribosome-recruitment mechanism yet discovered. By combining these findings with knowledge of canonical translation and the behavior of other IRESes, mechanistic models of this RNA structure-based process are emerging. PMID:18515544

  10. Targeting MicroRNA in Cancer Using Plant-Based Proanthocyanidins

    Directory of Open Access Journals (Sweden)

    Rishipal R. Bansode

    2016-04-01

    Full Text Available Proanthocyanidins are oligomeric flavonoids found in plant sources, most notably in apples, cinnamon, grape skin and cocoa beans. They have been also found in substantial amounts in cranberry, black currant, green tea, black tea and peanut skins. These compounds have been recently investigated for their health benefits. Proanthocyanidins have been demonstrated to have positive effects on various metabolic disorders such as inflammation, obesity, diabetes and insulin resistance. Another upcoming area of research that has gained widespread interest is microRNA (miRNA-based anticancer therapies. MicroRNAs are short non-coding RNA segments, which plays a crucial role in RNA silencing and post-transcriptional regulation of gene expression. Currently, miRNA based anticancer therapies are being investigated either alone or in combination with current treatment methods. In this review, we summarize the current knowledge and investigate the potential of naturally occurring proanthocyanidins in modulating miRNA expression. We will also assess the strategies and challenges of using this approach as potential cancer therapeutics.

  11. Shape codebook based handwritten and machine printed text zone extraction

    Science.gov (United States)

    Kumar, Jayant; Prasad, Rohit; Cao, Huiagu; Abd-Almageed, Wael; Doermann, David; Natarajan, Premkumar

    2011-01-01

    In this paper, we present a novel method for extracting handwritten and printed text zones from noisy document images with mixed content. We use Triple-Adjacent-Segment (TAS) based features which encode local shape characteristics of text in a consistent manner. We first construct two codebooks of the shape features extracted from a set of handwritten and printed text documents respectively. We then compute the normalized histogram of codewords for each segmented zone and use it to train a Support Vector Machine (SVM) classifier. The codebook based approach is robust to the background noise present in the image and TAS features are invariant to translation, scale and rotation of text. In experiments, we show that a pixel-weighted zone classification accuracy of 98% can be achieved for noisy Arabic documents. Further, we demonstrate the effectiveness of our method for document page classification and show that a high precision can be achieved for the detection of machine printed documents. The proposed method is robust to the size of zones, which may contain text content at line or paragraph level.

  12. Data Clustering Analysis Based on Wavelet Feature Extraction

    Institute of Scientific and Technical Information of China (English)

    QIANYuntao; TANGYuanyan

    2003-01-01

    A novel wavelet-based data clustering method is presented in this paper, which includes wavelet feature extraction and cluster growing algorithm. Wavelet transform can provide rich and diversified information for representing the global and local inherent structures of dataset. therefore, it is a very powerful tool for clustering feature extraction. As an unsupervised classification, the target of clustering analysis is dependent on the specific clustering criteria. Several criteria that should be con-sidered for general-purpose clustering algorithm are pro-posed. And the cluster growing algorithm is also con-structed to connect clustering criteria with wavelet fea-tures. Compared with other popular clustering methods,our clustering approach provides multi-resolution cluster-ing results,needs few prior parameters, correctly deals with irregularly shaped clusters, and is insensitive to noises and outliers. As this wavelet-based clustering method isaimed at solving two-dimensional data clustering prob-lem, for high-dimensional datasets, self-organizing mapand U-matrlx method are applied to transform them intotwo-dimensional Euclidean space, so that high-dimensional data clustering analysis,Results on some sim-ulated data and standard test data are reported to illus-trate the power of our method.

  13. Extracting Communities of Interests for Semantics-Based Graph Searches

    Science.gov (United States)

    Nakatsuji, Makoto; Tanaka, Akimichi; Uchiyama, Toshio; Fujimura, Ko

    Users recently find their interests by checking the contents published or mentioned by their immediate neighbors in social networking services. We propose semantics-based link navigation; links guide the active user to potential neighbors who may provide new interests. Our method first creates a graph that has users as nodes and shared interests as links. Then it divides the graph by link pruning to extract practical numbers, that the active user can navigate, of interest-sharing groups, i.e. communities of interests (COIs). It then attaches a different semantic tag to the link to each representative user, which best reflects the interests of COIs that they are included in, and to the link to each immediate neighbor of the active user. It finally calculates link attractiveness by analyzing the semantic tags on links. The active user can select the link to access by checking the semantic tags and link attractiveness. User interests extracted from large scale actual blog-entries are used to confirm the efficiency of our proposal. Results show that navigation based on link attractiveness and representative users allows the user to find new interests much more accurately than is otherwise possible.

  14. Object-based Analysis for Extraction of Dominant Tree Species

    Institute of Scientific and Technical Information of China (English)

    Meiyun; SHAO; Xia; JING; Lu; WANG

    2015-01-01

    As forest is of great significance for our whole development and the sustainable plan is so focus on it. It is very urgent for us to have the whole distribution,stock volume and other related information about that. So the forest inventory program is on our schedule. Aiming at dealing with the problem in extraction of dominant tree species,we tested the highly hot method-object-based analysis. Based on the ALOS image data,we combined multi-resolution in e Cognition software and fuzzy classification algorithm. Through analyzing the segmentation results,we basically extract the spruce,the pine,the birch and the oak of the study area. Both the spectral and spatial characteristics were derived from those objects,and with the help of GLCM,we got the differences of each species. We use confusion matrix to do the Classification accuracy assessment compared with the actual ground data and this method showed a comparatively good precision as 87% with the kappa coefficient 0. 837.

  15. Microfluidic droplet-based liquid-liquid extraction.

    Science.gov (United States)

    Mary, Pascaline; Studer, Vincent; Tabeling, Patrick

    2008-04-15

    We study microfluidic systems in which mass exchanges take place between moving water droplets, formed on-chip, and an external phase (octanol). Here, no chemical reaction takes place, and the mass exchanges are driven by a contrast in chemical potential between the dispersed and continuous phases. We analyze the case where the microfluidic droplets, occupying the entire width of the channel, extract a solute-fluorescein-from the external phase (extraction) and the opposite case, where droplets reject a solute-rhodamine-into the external phase (purification). Four flow configurations are investigated, based on straight or zigzag microchannels. Additionally to the experimental work, we performed two-dimensional numerical simulations. In the experiments, we analyze the influence of different parameters on the process (channel dimensions, fluid viscosities, flow rates, drop size, droplet spacing, ...). Several regimes are singled out. In agreement with the mass transfer theory of Young et al. (Young, W.; Pumir, A.; Pomeau, Y. Phys. Fluids A 1989, 1, 462), we find that, after a short transient, the amount of matter transferred across the droplet interface grows as the square root of time and the time it takes for the transfer process to be completed decreases as Pe-2/3, where Pe is the Peclet number based on droplet velocity and radius. The numerical simulation is found in excellent consistency with the experiment. In practice, the transfer time ranges between a fraction and a few seconds, which is much faster than conventional systems. PMID:18351786

  16. Probing electronic coupling between adenine bases in RNA strands from synchrotron radiation circular dichroism experiments

    DEFF Research Database (Denmark)

    Nielsen, Lisbeth Munksgård; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

    2012-01-01

    Circular dichroism spectra (176–330 nm) of RNA adenine oligomers, (rA)n (n = 1–10, 12, 15, and 20), reveal electronic coupling between two bases in short strands. The number of interacting bases in long strands is more and larger than that reported previously for the corresponding DNA strands....

  17. Modified gateway system for double shRNA expression and Cre/lox based gene expression

    Directory of Open Access Journals (Sweden)

    Leung Lisa

    2011-03-01

    Full Text Available Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2. Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  18. Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction of herbicides in peanuts.

    Science.gov (United States)

    Li, Na; Wang, Zhibing; Zhang, Liyuan; Nian, Li; Lei, Lei; Yang, Xiao; Zhang, Hanqi; Yu, Aimin

    2014-10-01

    Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction was developed and applied to the extraction of pesticides in high fatty matrices. The herbicides were ultrasonically extracted from peanut using ethyl acetate as extraction solvent. The separation of the analytes from a large amount of co-extractive fat was achieved by dispersive solid-phase extraction using MIL-101(Cr) as sorbent. In this step, the analytes were adsorbed on MIL-101(Cr) and the fat remained in bulk. The herbicides were separated and determined by high-performance liquid chromatography. The experimental parameters, including type and volume of extraction solvent, ultrasonication time, volume of hexane and eluting solvent, amount of MIL-101(Cr) and dispersive solid phase extraction time, were optimized. The limits of detection for herbicides range from 0.98 to 1.9 μg/kg. The recoveries of the herbicides are in the range of 89.5-102.7% and relative standard deviations are equal or lower than 7.0%. The proposed method is simple, effective and suitable for treatment of the samples containing high content of fat.

  19. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract

    International Nuclear Information System (INIS)

    The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • STAT3 directly regulates the levels of LncRNA HOTAIR. • LncRNA HOTAIR mediates the link between inflammation and EMT. • LncRNA HOTAIR is involved in the malignant transformation of cells caused by CSE

  20. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yi; Luo, Fei; Xu, Yuan; Wang, Bairu; Zhao, Yue; Xu, Wenchao; Shi, Le; Lu, Xiaolin; Liu, Qizhan, E-mail: drqzliu@hotmail.com

    2015-01-01

    The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • STAT3 directly regulates the levels of LncRNA HOTAIR. • LncRNA HOTAIR mediates the link between inflammation and EMT. • LncRNA HOTAIR is involved in the malignant transformation of cells caused by CSE.

  1. 三种方法提取血清miRNA效果的比较%Comparing the serum miRNA extraction effect of three methods

    Institute of Scientific and Technical Information of China (English)

    陈昌国; 陈秋圆; 郭建巍; 马志家; 刘敏; 赵强元; 张雅芳

    2015-01-01

    目的:比较Trizol法、蛋白酶K消化+ Trizol法及沉淀裂解法提取血清miRNA的效果。方法分别选取2例健康人、2例肺炎患者和2例肺癌患者血清,以Let‐7a‐5p作为目标mRNA ,线虫Cel‐miR‐39‐3p作为内参,分别采用Trizol法、蛋白酶K消化+ Trizol法及沉淀裂解法提取血清miRNA ,以Let‐7a‐5p及Cel‐miR‐39‐3p特异性引物进行反转录PCR ,以反转录PCR产物为模板运用SYBGreen Ⅰ法进行荧光定量PCR检测。结果 T rizol法与蛋白酶K消化+ Trizol法均能有效提取到血清miRNA ,而沉淀裂解法由于没有对血浆中的RNA进行富集故提取效果不佳。T rizol法提取较蛋白酶K消化+ T rizol法相比,可以减少蛋白酶K的消化时间且提取效果更好,并且血清与Trizol试剂的体积比为200∶600时效果相对较好。三种方法检测Cel‐miR‐39‐3p的Ct值基本一致。Tr‐izol法和蛋白酶K消化+ Trizol法在提取过程中加入的内参丢失较少。结论 Trizol法作为总RNA提取的手段适用于血清miRNA提取,通过调整血清与Trizol试剂的用量可使提取到的血清miRNA能够满足试验需要。%Objective To compare the serum miRNA extracting efficiency of Trizol method ,protease K diges‐tion combined with Trizol method and precipitation pyrolysis method .Methods 2 healthy persons ,2 pneumonia pa‐tients and 2 lung cancer patients were enrolled in the study and their serum samples were collected .miRNA were ex‐tracted from serum samples respectively by using Trizol method ,protease K digestion combined with Trizol method and precipitation pyrolysis method .Let‐7a‐5p was the objective miRNA of detection ,with Cel‐miR‐39‐3p as the incor‐poration of spike‐in reference .The specific primers of Let‐7a‐5p (objective miRNA) and Cel‐miR‐39‐3p (internal ref‐erence) were used in reverse transcription PCR .The products of reverse transcription PCR were used as

  2. Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.

    Directory of Open Access Journals (Sweden)

    Satoshi Iizuka

    Full Text Available Head and neck squamous cell carcinoma (HNSCC exhibits increased expression of cyclin D1 (CCND1. Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA. In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.

  3. Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.

    Science.gov (United States)

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs. PMID:25437003

  4. An Improved Method for the Extraction of Total RNA from Spruce Needles%一种改良的云杉针叶总RNA提取方法

    Institute of Scientific and Technical Information of China (English)

    刘洁; 江静怡; 徐吉臣

    2014-01-01

    云杉(Picea)是一种重要的绿化和用材树种。根据云杉的特点,本研究提出了改良的云杉RNA提取方法即SDS酚-LiCl法,以不同种云杉(青杄,白杄和红皮)的针叶为试验材料,采用TRIzol法、CTAB法、改进的SDS酚法和SDS酚-LiCl法提取RNA,并进行了质量验证。结果表明:TRIzol法不能提取云杉针叶总RNA;CTAB法和改进的SDS酚法提取的RNA质量少,并存在不同程度的多糖、多酚和DNA污染。 SDS酚-LiCl法适当增加了提取液中SDS、PVP以及β-巯基乙醇的浓度,结合酸性条件、高盐溶液(3 mol/L NaAc)和LiCl等减少DNA、多糖污染的步骤,最终提取的RNA质量好、得率高。进一步反转录后,利用特异引物进行扩增验证,发现扩增的片段条带清晰,得率高,片段大小符合预期。研究结果表明,SDS酚-LiCl法可以较好的提取云杉RNA并进行后续的基因表达及转录组研究,为推动云杉在转录水平上的研究奠定了基础。%Spruce (Picea) is one of the important virescence and timber species. According to the characteristics of spruce, we improved a new RNA extraction method called SDS phenol-LiCl. Needles of three spruce species Picea wilsonii, Picea meyeri and Picea koraiensis were used as experiment materials for RNA extraction. The RNA extracting effects of SDS phenol-LiCl method and the previous TRIzol, CTAB, improved SDS-phenol methods were analyzed and compared. The results showed that TRIzol method failed for RNA extraction from spruce. CTAB and improved SDS-phenol methods could be used to extract spruce RNA but with less amount and contamination of polysaccharides, polyphenol and DNA. With increased addition of SDS, PVP and β-mercap-toethanol, and combined use of acidic condition, high concentration of salt (3 mol/L NaAc) and LiCl to decrease the DNA and polysaccharides contamination, the spruce RNA extracted by SDS phenol-LiCl method was in high quality and yield. The reverse

  5. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies

    KAUST Repository

    Chawla, Mohit

    2015-06-27

    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.

  6. Optimization of RNA-based c-di-GMP fluorescent sensors through tuning their structural modules.

    Science.gov (United States)

    Inuzuka, Saki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

    2016-08-01

    Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors.

  7. URS DataBase: universe of RNA structures and their motifs.

    Science.gov (United States)

    Baulin, Eugene; Yacovlev, Victor; Khachko, Denis; Spirin, Sergei; Roytberg, Mikhail

    2016-01-01

    The Universe of RNA Structures DataBase (URSDB) stores information obtained from all RNA-containing PDB entries (2935 entries in October 2015). The content of the database is updated regularly. The database consists of 51 tables containing indexed data on various elements of the RNA structures. The database provides a web interface allowing user to select a subset of structures with desired features and to obtain various statistical data for a selected subset of structures or for all structures. In particular, one can easily obtain statistics on geometric parameters of base pairs, on structural motifs (stems, loops, etc.) or on different types of pseudoknots. The user can also view and get information on an individual structure or its selected parts, e.g. RNA-protein hydrogen bonds. URSDB employs a new original definition of loops in RNA structures. That definition fits both pseudoknot-free and pseudoknotted secondary structures and coincides with the classical definition in case of pseudoknot-free structures. To our knowledge, URSDB is the first database supporting searches based on topological classification of pseudoknots and on extended loop classification.Database URL: http://server3.lpm.org.ru/urs/.

  8. 少白细胞血小板miRNA提取方法*%Method of extraction of miRNA from human leucocyte depleted-platelet

    Institute of Scientific and Technical Information of China (English)

    罗茂; 万沁; 刘飞; 马卫中; 吴剑波

    2013-01-01

    Objective: To establish a simple and convenient method isolation of high-quality miRNA from human leukocyte-depleted platelet preparation. Methods: Small RNA in healthy platelets was extracted by conv-entional gradient centrifugation, MACS and the mirVanaTM miRNA Isolation Kit. Leukocyte contamination in the final purified platelet preparation was estimated by conventional gradient centrifugation, platelet counting and the transcript levels of the gene encoding CD45/GPIIb marker in platelet preparations. The concentration, purity and integrity were detected, and a miRNA' cDNA library which was constructed by RNA-tailing and primer-extension reverse transcription (RT)-PCR was used as the template for the amplification of U6 and miR-449b to verify the authenticity. Results:Small RNA was extracted with high purity, high efficiency of leukocyte depletion, Its cDNA had high authenticity despite its low concentration. Conclusion:A method of extraction of microRNA from human platelet preparation with high efficiency of leukocyte depletion, and high quality of miRNA' cDNA library is constructed, which is applicative to future miRNA experiments.%  目的:建立常规梯度离心结合MACS磁珠分选纯化和小分子RNA分离试剂盒简便快速提取血小板miRNA的方法。方法:利用常规梯度离心获取血小板后,结合MACS磁珠分选和小分子RNA分离试剂盒提取少白细胞血小板(LDP)小分子RNA;经血小板计数、CD45和GPIIb mRNA marker PCR扩增检测,判定纯化效果;通过小分子RNA浓度、纯度及毛细管电泳完整性检测,以及内参U6和血小板miR-449b加尾及引物延伸RT-PCR后PCR扩增结果验证构建血小板miRNA的cDNA模板真实性。结果:提取小分子RNA纯度较高,有效去除白细胞污染,虽然含量较少,但内参U6和血小板miR-449b真实性验证结果提示,构建血小板miRNA的cDNA模板质量较高。结论:常规梯度离心结合MACS磁珠分选纯化和小分子RNA

  9. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry.

    Science.gov (United States)

    Carter, Charles W; Wolfenden, Richard

    2016-01-01

    The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology.

  10. Sensitive voltammetric detection of yeast RNA based on its interaction with Victoria Blue B

    Directory of Open Access Journals (Sweden)

    WEI SUN

    2009-12-01

    Full Text Available Voltammetric studies of the interaction of yeast RNA (y-RNA with Victoria Blue B (VBB are described in this paper. Furthermore, a linear sweep voltammetric method for the detection of y-RNA was established. The reaction conditions, such as acidity and amount of buffer solution, the concentration of VBB, the reaction time and temperature, etc., were carefully investigated by second order derivative linear sweep voltammetry. Under the optimal conditions, the reduction peak current of VBB at –0.75 V decreased greatly after the addition of y-RNA to the solution without any shift of the reduction peak potential. Based on the decrease of the peak current, a new quantitative method for the determination of y-RNA was developed. The effects of co-existing substances on the determination were carefully investigated and three synthetic samples were determined with satisfactory results. The stoichiometry of the VBB–y-RNA complex was calculated by linear sweep voltammetry and the interaction mechanism is discussed.

  11. Cell-free protein expression based on extracts from CHO cells.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the

  12. Vision-Based Faint Vibration Extraction Using Singular Value Decomposition

    Directory of Open Access Journals (Sweden)

    Xiujun Lei

    2015-01-01

    Full Text Available Vibration measurement is important for understanding the behavior of engineering structures. Unlike conventional contact-type measurements, vision-based methodologies have attracted a great deal of attention because of the advantages of remote measurement, nonintrusive characteristic, and no mass introduction. It is a new type of displacement sensor which is convenient and reliable. This study introduces the singular value decomposition (SVD methods for video image processing and presents a vibration-extracted algorithm. The algorithms can successfully realize noncontact displacement measurements without undesirable influence to the structure behavior. SVD-based algorithm decomposes a matrix combined with the former frames to obtain a set of orthonormal image bases while the projections of all video frames on the basis describe the vibration information. By means of simulation, the parameters selection of SVD-based algorithm is discussed in detail. To validate the algorithm performance in practice, sinusoidal motion tests are performed. Results indicate that the proposed technique can provide fairly accurate displacement measurement. Moreover, a sound barrier experiment showing how the high-speed rail trains affect the sound barrier nearby is carried out. It is for the first time to be realized at home and abroad due to the challenge of measuring environment.

  13. COSMO-RS-based extractant screening for phenol extraction as model system

    NARCIS (Netherlands)

    Burghoff, B.; Goetheer, E.L.V.; Haan, A.B. de

    2008-01-01

    The focus of this investigation is the development of a fast and reliable extractant screening approach. Phenol extraction is selected as the model process. A quantum chemical conductor-like screening model for real solvents (COSMO-RS) is combined with molecular design considerations. For this purpo

  14. An image segmentation based method for iris feature extraction

    Institute of Scientific and Technical Information of China (English)

    XU Guang-zhu; ZHANG Zai-feng; MA Yi-de

    2008-01-01

    In this article, the local anomalistic blocks such ascrypts, furrows, and so on in the iris are initially used directly asiris features. A novel image segmentation method based onintersecting cortical model (ICM) neural network was introducedto segment these anomalistic blocks. First, the normalized irisimage was put into ICM neural network after enhancement.Second, the iris features were segmented out perfectly and wereoutput in binary image type by the ICM neural network. Finally,the fourth output pulse image produced by ICM neural networkwas chosen as the iris code for the convenience of real timeprocessing. To estimate the performance of the presentedmethod, an iris recognition platform was produced and theHamming Distance between two iris codes was computed tomeasure the dissimilarity between them. The experimentalresults in CASIA vl.0 and Bath iris image databases show thatthe proposed iris feature extraction algorithm has promisingpotential in iris recognition.

  15. Automated Smiley Face Extraction Based on Genetic Algorithm

    Directory of Open Access Journals (Sweden)

    Md Alamgir Hossain

    2012-07-01

    Full Text Available Facial expression scrutiny has attracted tremendous consciousness in the area of computer vision because it plays a prime role in the domain of human-machine communication. Smiley face expressions are generated by slimming down of facial muscles, which results in temporally buckled facial features such as eye lids, eye brows, nose, lips, and skin texture. These are evaluated by three characteristics: those portions of the face that will take part for facial action, the intensity of facial actions, and the dynamics of facial actions. In this paper we propose a real-time, accurate, and robust smile detection method based on genetic algorithm. We generated leaf-matrix to extract target expression. Finally, we have compared our methodology with the smile shutter function of Canon Camera. We have achieved better performance than Sony on slight smile.

  16. Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Ercan Selçuk Ünlü

    Full Text Available Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

  17. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    Science.gov (United States)

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. PMID:18533829

  18. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

    Directory of Open Access Journals (Sweden)

    Roh C

    2012-05-01

    Full Text Available Changhyun RohDivision of Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Jeongeup, Republic of KoreaAbstract: Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS, and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV nucleocapsid (N protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (--catechin gallate and (--gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (--catechin gallate and (--gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 µg mL–1, (--catechin gallate and (--gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.Keywords: SARS, RNA oligonucleotide, quantum dots, inhibitor, screening

  19. incaRNAfbinv: a web server for the fragment-based design of RNA sequences.

    Science.gov (United States)

    Drory Retwitzer, Matan; Reinharz, Vladimir; Ponty, Yann; Waldispühl, Jérôme; Barash, Danny

    2016-07-01

    In recent years, new methods for computational RNA design have been developed and applied to various problems in synthetic biology and nanotechnology. Lately, there is considerable interest in incorporating essential biological information when solving the inverse RNA folding problem. Correspondingly, RNAfbinv aims at including biologically meaningful constraints and is the only program to-date that performs a fragment-based design of RNA sequences. In doing so it allows the design of sequences that do not necessarily exactly fold into the target, as long as the overall coarse-grained tree graph shape is preserved. Augmented by the weighted sampling algorithm of incaRNAtion, our web server called incaRNAfbinv implements the method devised in RNAfbinv and offers an interactive environment for the inverse folding of RNA using a fragment-based design approach. It takes as input: a target RNA secondary structure; optional sequence and motif constraints; optional target minimum free energy, neutrality and GC content. In addition to the design of synthetic regulatory sequences, it can be used as a pre-processing step for the detection of novel natural occurring RNAs. The two complementary methodologies RNAfbinv and incaRNAtion are merged together and fully implemented in our web server incaRNAfbinv, available at http://www.cs.bgu.ac.il/incaRNAfbinv. PMID:27185893

  20. Technological development of structural DNA/RNA-based RNAi systems and their applications.

    Science.gov (United States)

    Jeong, Eun Hye; Kim, Hyejin; Jang, Bora; Cho, Hyesoo; Ryu, Jaehee; Kim, Boyeon; Park, Youngkuk; Kim, Jieun; Lee, Jong Bum; Lee, Hyukjin

    2016-09-01

    RNA interference (RNAi)-based gene therapy has drawn tremendous attention due to its highly specific gene regulation by selective degradation of any target mRNA. There have been multiple reports regarding the development of various cationic materials for efficient siRNA delivery, however, many studies still suffer from the conventional delivery problems such as suboptimal transfection performance, a lack of tissue specificity, and potential cytotoxicity. Despite the huge therapeutic potential of siRNAs, conventional gene carriers have failed to guarantee successful gene silencing in vivo, thus not warranting clinical trials. The relatively short double-stranded structure of siRNAs has resulted in uncompromising delivery formulations, as well as low transfection efficiency, compared with the conventional nucleic acid drugs such as plasmid DNAs. Recent developments in structural siRNA and RNAi nanotechnology have enabled more refined and reliable in vivo gene silencing with multiple advantages over naked siRNAs. This review focuses on recent progress in the development of structural DNA/RNA-based RNAi systems and their potential therapeutic applications. In addition, an extensive list of prior reports on various RNAi systems is provided and categorized by their distinctive molecular characters. PMID:26494399

  1. Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus.

    OpenAIRE

    Pacini, D L; Brady, M T; Budde, C T; Connell, M J; Hamparian, V V; Hughes, J. H.

    1988-01-01

    Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE we...

  2. Mechanism of gold solvent extraction from aurocyanide solution by quaternary amines: models of extracting species based on hydrogen bonding

    Institute of Scientific and Technical Information of China (English)

    马刚; 闫文飞; 陈景; 严纯华; 高宏成; 周维金; 施鼐; 吴谨光; 徐光宪; 黄昆; 余建民; 崔宁

    2000-01-01

    The mechanism of gold solvent extraction from KAu(CN)2 solution was investigated by means of FTIR, EXAFS, ICP and radioactive tracer methods. Two extraction systems were studied, namely N263-tributyl phosphate(TBP)-n-dodecane and N263-iso-octanol-n-dodecane. High-resolution FT IR spectroscopy indicated that the CN stretching vibrations of the two extraction systems differred greatly. In order to interpret the significant difference in CN stretching vibrations, twoextracting species models are proposed——supramolecular structures based on the formation ofhydrogen bonds between Au(CN)2- and modifiers such as TBP and iso-octanol.

  3. Improved CTAB Method for Total RNA Extraction of Mature Feicheng Peach Fruit%改良CTAB法提取成熟肥城桃果实的总RNA

    Institute of Scientific and Technical Information of China (English)

    陈长宝; 朱树华; 周杰

    2009-01-01

    为了从成熟的肥城桃果实中提取纯净完整的RNA,本试验在原CTAB法的基础上进行了改进,并对RNA的完整性、产率和纯度等方面进行评价.结果表明,采用改良CTAB法所得RNA完整性好,条带清晰,无明显降解,28S和18S rRNA的亮度比约为2∶1,A260/A280达1.89,且产率较高,为356.21 μg/g,经RT-PCR后得到一特异条带,表明该RNA可用于后续分子生物学操作.

  4. 肝癌石蜡包埋组织miRNA提取方法的优化%Optimization of MicroRNA extraction method from HCC FFPE tissues

    Institute of Scientific and Technical Information of China (English)

    党裔武; 容敏华; 陈罡

    2012-01-01

    Objective To modify and optimize the microRNA(miRNA) extraction and detection approach from hepatocellular carcinoma(HCC) formalin fixed paraffin embedded(FFPE) tissues. Methods miRNeasy FFPE kit was performed to investigate the optimizational thickness oi sections from HCC FFPE tissues and. time of protea.se K. treatment. Afterwards,expression of miR. 221, miR 146a,let7 a,miR 191,miR 103 and RNU6B was detected by real time RT PCR. The difference of the miRNA expression be tween fresh cells and FFPE tissues from the same cell line was also compared by real time RT PCR. Results 30 μm thickness and 48 h of proteinase K treatment were the best parameters to isolate miRNA. Different expression levels of all the miRNAs could be detected in both the HCC cells samples and clinic FFPE samples. There was no significant variation of the miRNA expression be tween fresh cells and FFPE samples from the same cell lines. Conclusion The optimizational miRNeasy FFPE method is applicable for miRNA extraction from FFPE tissues. The method is productive and can be popularized for the miRNA isolation for different FFPE tissues.%目的 改进并优化从肝癌(HCC)甲醛固定石蜡包埋组织(FFPE)提取并检测微小RNA(miRNA)的方法.方法 使用miRNeasy FFPE试剂盒探讨提取HCC石蜡组织miRNA最佳切片厚度及蛋白酶K作用时间.实时定量RT-PCR检测miR-221、miR-146a、let7-a、miR-191、miR-103及RNU6B的表达量.将HCC细胞制成石蜡组织,对比同一细胞系新鲜细胞及石蜡包埋细胞的各个miRNA表达量差异.结果 切片厚度30 μm,蛋白酶K作用时间48 h时提取miRNA效果最佳.各HCC细胞系及临床HCC石蜡组织均有不同水平的miRNA-221、miR-146a、let7-a、miR-191、miR-103及RNU6B表达.同一细胞系新鲜细胞与石蜡包埋后的各miRNA表达无明显差别.结论 改良后的miRNeasy FFPE 提取方法能提取石蜡组织中的miRNA,可重复性强,可推广用于临床各种石蜡包埋组织的miRNA提取.

  5. Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte;

    2015-01-01

    by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S, ACTB, IL1B, IL1RN, IL1R2, and PGK1 using qPCR. Results: The MagMAX system extracted 2.3-2.8 times more RNA per mL blood, with better performance...... in terms of purity, and with comparable levels of integrity relative to the PAXgene system. Gene expression analysis using qPCR of 18S, ACTB, IL1B, IL1RN, IL1R2, and the promising blood-specific reference gene, PGK1, revealed negligible differences (

  6. Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Garwick-Coppens Sara E

    2011-11-01

    Full Text Available Abstract Background RNA interference (RNAi is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs. Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. Results As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. Conclusions We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

  7. A mRNA-Responsive G-Quadruplex-Based Drug Release System

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2015-04-01

    Full Text Available G-quadruplex-based drug delivery carriers (GDDCs were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II phthalocyanine (CuAPC. As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR mRNA. Structural analysis using circular dichroism (CD spectroscopy showed that the GDDCs form a (3 + 1 type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

  8. Novel zinc-based fixative for high quality DNA, RNA and protein analysis

    OpenAIRE

    Lykidis, Dimitrios; Van Noorden, Susan; Armstrong, Alan; Spencer-Dene, Bradley; Li, Jie; Zhuang, Zhengping; Stamp, Gordon W. H.

    2007-01-01

    We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT–PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was signific...

  9. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Directory of Open Access Journals (Sweden)

    Anja Gulliksen

    2012-01-01

    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  10. Energy Based Feature Extraction for Classification of Respiratory Signals Using Modified Threshold Based Algorithm

    Directory of Open Access Journals (Sweden)

    A.BHAVANI SANKAR,

    2010-10-01

    Full Text Available In this work, we carried out a detailed study of various features of respiratory signal. Respiratory signals contains potentially precise information that could assist clinicians in making appropriate and timely decisions during sleeping disorder and labour. The extraction and detection of the sleep apnea from composite abdominal signals with powerful and advance methodologies is becoming a very important requirement in apnea patient monitoring. The method we proposed in this work is based on the extraction of four main features of respiratory signal. The automatic signal classification starts by extracting signal features from 30 seconds respiratory data through autoregressive modeling (AR and other techniques. Four features are: signal energy, zero crossing frequency, dominant frequency estimated by AR and strength of dominant frequency based on AR. These features are then compared to threshold values and introduced to a series of conditions to determine the signal category for each specific epoch.

  11. A plasmonic colorimetric strategy for visual miRNA detection based on hybridization chain reaction.

    Science.gov (United States)

    Miao, Jie; Wang, Jingsheng; Guo, Jinyang; Gao, Huiguang; Han, Kun; Jiang, Chengmin; Miao, Peng

    2016-01-01

    In this work, a novel colorimetric strategy for miRNA analysis is proposed based on hybridization chain reaction (HCR)-mediated localized surface plasmon resonance (LSPR) variation of silver nanoparticles (AgNPs). miRNA in the sample to be tested is able to release HCR initiator from a solid interface to AgNPs colloid system by toehold exchange-mediated strand displacement, which then triggers the consumption of fuel strands with single-stranded tails for HCR. The final produced long nicked double-stranded DNA loses the ability to protect AgNPs from salt-induced aggregation. The stability variation of the colloid system can then be monitored by recording corresponding UV-vis spectrum and initial miRNA level is thus determined. This sensing system involves only four DNA strands which is quite simple. The practical utility is confirmed to be excellent by employing different biological samples. PMID:27534372

  12. A Novel Framework Based on ACO and PSO for RNA Secondary Structure Prediction

    Directory of Open Access Journals (Sweden)

    Gang Wang

    2013-01-01

    Full Text Available Prediction of RNA structure is a useful process for creating new drugs and understanding genetic diseases. In this paper, we proposed a particle swarm optimization (PSO and ant colony optimization (ACO based framework (PAF for RNA secondary structure prediction. PAF consists of crucial stem searching (CSS and global sequence building (GSB. In CSS, a modified ACO (MACO is used to search the crucial stems, and then a set of stems are generated. In GSB, we used a modified PSO (MPSO to construct all the stems in one sequence. We evaluated the performance of PAF on ten sequences, which have length from 122 to 1494. We also compared the performance of PAF with the results obtained from six existing well-known methods, SARNA-Predict, RnaPredict, ACRNA, PSOfold, IPSO, and mfold. The comparison results show that PAF could not only predict structures with higher accuracy rate but also find crucial stems.

  13. Analysis on the Physicochemical Properties of Ginkgo biloba Leaves after Enzymolysis Based Ultrasound Extraction and Soxhlet Extraction

    Directory of Open Access Journals (Sweden)

    Chang-Wei Zhang

    2016-01-01

    Full Text Available In this study, high performance liquid chromatography (HPLC, ultraviolet (UV, thermagravimetric analyzer (TGA, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS, and scanning electron microscope (SEM were used as measurement techniques, contents of chemical composition, pyrolytic products, thermal stability, morphological characterization of Ginkgo biloba leaves (GBL acted as the index, and physicochemical properties of GBL after enzymolysis based ultrasound extraction (EBUE and Soxhlet extraction were studied. The detection results of chemical composition revealed that contents of general flavone, soluble protein, soluble total sugar and protein in the GBL declined significantly after EBUE, and contents of polyprenols and crude fat obviously reduced as well after Soxhlet extraction. Py-GC-MS results indicated that total GC contents of micromolecules with carbon less than 12 from 54.0% before EBUE decline to 8.34% after EBUE. Total GC contents of long-chain fatty acids with carbon less than 20 from 43.0% before EBUE reduced to 27.0% after Soxhlet extraction. Thermal stability results showed that GBL after Soxhlet extraction was easier to decompose than GBL before EBUE. SEM results illustrated that surface structure of GBL was damaged severely after EBUE, compared with GBL before EBUE, while organic solvent extraction had little influence on the morphological characterization of GBL after Soxhlet extraction compared with GBL after EBUE.

  14. Analysis on the Physicochemical Properties of Ginkgo biloba Leaves after Enzymolysis Based Ultrasound Extraction and Soxhlet Extraction.

    Science.gov (United States)

    Zhang, Chang-Wei; Wang, Cheng-Zhang; Tao, Ran

    2016-01-15

    In this study, high performance liquid chromatography (HPLC), ultraviolet (UV), thermagravimetric analyzer (TGA), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), and scanning electron microscope (SEM) were used as measurement techniques, contents of chemical composition, pyrolytic products, thermal stability, morphological characterization of Ginkgo biloba leaves (GBL) acted as the index, and physicochemical properties of GBL after enzymolysis based ultrasound extraction (EBUE) and Soxhlet extraction were studied. The detection results of chemical composition revealed that contents of general flavone, soluble protein, soluble total sugar and protein in the GBL declined significantly after EBUE, and contents of polyprenols and crude fat obviously reduced as well after Soxhlet extraction. Py-GC-MS results indicated that total GC contents of micromolecules with carbon less than 12 from 54.0% before EBUE decline to 8.34% after EBUE. Total GC contents of long-chain fatty acids with carbon less than 20 from 43.0% before EBUE reduced to 27.0% after Soxhlet extraction. Thermal stability results showed that GBL after Soxhlet extraction was easier to decompose than GBL before EBUE. SEM results illustrated that surface structure of GBL was damaged severely after EBUE, compared with GBL before EBUE, while organic solvent extraction had little influence on the morphological characterization of GBL after Soxhlet extraction compared with GBL after EBUE.

  15. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    Science.gov (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  16. A permutation based simulated annealing algorithm to predict pseudoknotted RNA secondary structures.

    Science.gov (United States)

    Tsang, Herbert H; Wiese, Kay C

    2015-01-01

    Pseudoknots are RNA tertiary structures which perform essential biological functions. This paper discusses SARNA-Predict-pk, a RNA pseudoknotted secondary structure prediction algorithm based on Simulated Annealing (SA). The research presented here extends previous work of SARNA-Predict and further examines the effect of the new algorithm to include prediction of RNA secondary structure with pseudoknots. An evaluation of the performance of SARNA-Predict-pk in terms of prediction accuracy is made via comparison with several state-of-the-art prediction algorithms using 20 individual known structures from seven RNA classes. We measured the sensitivity and specificity of nine prediction algorithms. Three of these are dynamic programming algorithms: Pseudoknot (pknotsRE), NUPACK, and pknotsRG-mfe. One is using the statistical clustering approach: Sfold and the other five are heuristic algorithms: SARNA-Predict-pk, ILM, STAR, IPknot and HotKnots algorithms. The results presented in this paper demonstrate that SARNA-Predict-pk can out-perform other state-of-the-art algorithms in terms of prediction accuracy. This supports the use of the proposed method on pseudoknotted RNA secondary structure prediction of other known structures. PMID:26558299

  17. Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes.

    Directory of Open Access Journals (Sweden)

    Christopher A Lavender

    2015-05-01

    Full Text Available HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively. Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.

  18. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection. PMID:27283884

  19. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  20. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    Science.gov (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  1. A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA

    OpenAIRE

    Onizuka, Kazumitsu; Taniguchi, Yosuke; Sasaki, Shigeki

    2010-01-01

    Site-specific modification of RNA is of great significance to investigate RNA structure, function and dynamics. Recently, we reported a new method for sequence- and cytosine-selective chemical modification of RNA based on the functional group transfer reaction of the 1-phenyl-2-methylydene-1,3-diketone unit of the 6-thioguanosine base incorporated in the oligodeoxynucleotide probe. In this study, we describe that the functionality transfer rate is greatly enhanced and the selectivity is shift...

  2. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

    Directory of Open Access Journals (Sweden)

    Maria Greco

    Full Text Available The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11 isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5 which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols. The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1 fresh weigh of RNA and DNA, respectively and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates

  3. PCR-based typing of DNA extracted from cigarette butts.

    Science.gov (United States)

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  4. STATISTICAL BASED NON-LINEAR MODEL UPDATING USING FEATURE EXTRACTION

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, J.F.; Hemez, F.M. [and others

    2000-10-01

    This research presents a new method to improve analytical model fidelity for non-linear systems. The approach investigates several mechanisms to assist the analyst in updating an analytical model based on experimental data and statistical analysis of parameter effects. The first is a new approach at data reduction called feature extraction. This is an expansion of the update metrics to include specific phenomena or character of the response that is critical to model application. This is an extension of the classical linear updating paradigm of utilizing the eigen-parameters or FRFs to include such devices as peak acceleration, time of arrival or standard deviation of model error. The next expansion of the updating process is the inclusion of statistical based parameter analysis to quantify the effects of uncertain or significant effect parameters in the construction of a meta-model. This provides indicators of the statistical variation associated with parameters as well as confidence intervals on the coefficients of the resulting meta-model, Also included in this method is the investigation of linear parameter effect screening using a partial factorial variable array for simulation. This is intended to aid the analyst in eliminating from the investigation the parameters that do not have a significant variation effect on the feature metric, Finally an investigation of the model to replicate the measured response variation is examined.

  5. CEMS using hot wet extractive method based on DOAS

    Science.gov (United States)

    Sun, Bo; Zhang, Chi; Sun, Changku

    2011-11-01

    A continuous emission monitoring system (CEMS) using hot wet extractive method based on differential optical absorption spectroscopy (DOAS) is designed. The developed system is applied to retrieving the concentration of SO2 and NOx in flue gas on-site. The flue gas is carried along a heated sample line into the sample pool at a constant temperature above the dew point. In this case, the adverse impact of water vapor on measurement accuracy is reduced greatly, and the on-line calibration is implemented. And then the flue gas is discharged from the sample pool after the measuring process is complete. The on-site applicability of the system is enhanced by using Programmable Logic Controller (PLC) to control each valve in the system during the measuring and on-line calibration process. The concentration retrieving method used in the system is based on the partial least squares (PLS) regression nonlinear method. The relationship between the known concentration and the differential absorption feature gathered by the PLS nonlinear method can be figured out after the on-line calibration process. Then the concentration measurement of SO2 and NOx can be easily implemented according to the definite relationship. The concentration retrieving method can identify the information and noise effectively, which improves the measuring accuracy of the system. SO2 with four different concentrations are measured by the system under laboratory conditions. The results proved that the full-scale error of this system is less than 2%FS.

  6. An Automated Video Object Extraction System Based on Spatiotemporal Independent Component Analysis and Multiscale Segmentation

    Directory of Open Access Journals (Sweden)

    Zhang Xiao-Ping

    2006-01-01

    Full Text Available Video content analysis is essential for efficient and intelligent utilizations of vast multimedia databases over the Internet. In video sequences, object-based extraction techniques are important for content-based video processing in many applications. In this paper, a novel technique is developed to extract objects from video sequences based on spatiotemporal independent component analysis (stICA and multiscale analysis. The stICA is used to extract the preliminary source images containing moving objects in video sequences. The source image data obtained after stICA analysis are further processed using wavelet-based multiscale image segmentation and region detection techniques to improve the accuracy of the extracted object. An automated video object extraction system is developed based on these new techniques. Preliminary results demonstrate great potential for the new stICA and multiscale-segmentation-based object extraction system in content-based video processing applications.

  7. COASTLINE EXTRACTION FROM AERIAL IMAGES BASED ON EDGE DETECTION

    OpenAIRE

    Paravolidakis, V.; Moirogiorgou, K.; Ragia, L.; Zervakis, M.; Synolakis, C.

    2016-01-01

    Nowadays coastline extraction and tracking of its changes become of high importance because of the climate change, global warming and rapid growth of human population. Coastal areas play a significant role for the economy of the entire region. In this paper we propose a new methodology for automatic extraction of the coastline using aerial images. A combination of a four step algorithm is used to extract the coastline in a robust and generalizable way. First, noise distortion is reduced in or...

  8. The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

    Directory of Open Access Journals (Sweden)

    Nicholas A Kennedy

    Full Text Available Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories.Fecal samples from two healthy volunteers (H3 and H4 and two relapsing IBD patients (I1 and I2 were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient.DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561 and 22 (IQR 9-36 ng/µL respectively (p<0.0001. Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05.This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring

  9. Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

    Directory of Open Access Journals (Sweden)

    Rie Kawai

    2012-03-01

    Full Text Available Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienylimidazo[4,5-b]pyridine (Ds and pyrrole-2-carbaldehyde (Pa functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

  10. Chronic oral administration of pine bark extract (flavangenol) attenuates brain and liver mRNA expressions of HSPs in heat-exposed chicks.

    Science.gov (United States)

    Yang, Hui; Chowdhury, Vishwajit S; Bahry, Mohammad A; Tran, Phuong V; Do, Phong H; Han, Guofeng; Zhang, Rong; Tagashira, Hideki; Tsubata, Masahito; Furuse, Mitsuhiro

    2016-08-01

    Exposure to a high ambient temperature (HT) can cause heat stress, which has a huge negative impact on physiological functions. Cellular heat-shock response is activated upon exposure to HT for cellular maintenance and adaptation. In addition, antioxidants are used to support physiological functions under HT in a variety of organisms. Flavangenol, an extract of pine bark, is one of the most potent antioxidants with its complex mixture of polyphenols. In the current study, chronic (a single daily oral administration for 14 days) or acute (a single oral administration) oral administration of flavangenol was performed on chicks. Then the chicks were exposed to an acute HT (40±1°C for 3h) to examine the effect of flavangenol on the mRNA expression of heat-shock protein (HSP) in the brain and liver. Rectal temperature, plasma aspartate aminotransferase (AAT), a marker of liver damage, and plasma corticosterone as well as metabolites were also determined. HSP-70 and -90 mRNA expression, rectal temperature, plasma AAT and corticosterone were increased by HT. Interestingly, the chronic, but not the acute, administration of flavangenol caused a declining in the diencephalic mRNA expression of HSP-70 and -90 and plasma AAT in HT-exposed chicks. Moreover, the hepatic mRNA expression of HSP-90 was also significantly decreased by chronic oral administration of flavangenol in HT chicks. These results indicate that chronic, but not acute, oral administration of flavangenol attenuates HSP mRNA expression in the central and peripheral tissues due to its possible role in improving cellular protective functions during heat stress. The flavangenol-dependent decline in plasma AAT further suggests that liver damage induced by heat stress was minimized by flavangenol.

  11. Fluorescence-based detection of single-nucleotide changes in RNA using graphene oxide and DNAzyme.

    Science.gov (United States)

    Hong, Chaesun; Kim, Dong-Min; Baek, Ahruem; Chung, Hyewon; Jung, Woong; Kim, Dong-Eun

    2015-04-01

    We report a simple fluorometric method for detection of single-nucleotide changes in RNA using graphene oxide (GO) and RNA-cleaving DNAzyme. The fluorescent DNA probe (F-DNA) was annealed to RNA fragments generated by RNA cleavage with DNAzyme specific to mutant RNA. The F-DNA-RNA duplex attenuated the quenching of F-DNA fluorescence by GO. PMID:25714982

  12. A quantitative analysis of secondary RNA structure using domination based parameters on trees

    Directory of Open Access Journals (Sweden)

    Zou Yue

    2006-03-01

    Full Text Available Abstract Background It has become increasingly apparent that a comprehensive database of RNA motifs is essential in order to achieve new goals in genomic and proteomic research. Secondary RNA structures have frequently been represented by various modeling methods as graph-theoretic trees. Using graph theory as a modeling tool allows the vast resources of graphical invariants to be utilized to numerically identify secondary RNA motifs. The domination number of a graph is a graphical invariant that is sensitive to even a slight change in the structure of a tree. The invariants selected in this study are variations of the domination number of a graph. These graphical invariants are partitioned into two classes, and we define two parameters based on each of these classes. These parameters are calculated for all small order trees and a statistical analysis of the resulting data is conducted to determine if the values of these parameters can be utilized to identify which trees of orders seven and eight are RNA-like in structure. Results The statistical analysis shows that the domination based parameters correctly distinguish between the trees that represent native structures and those that are not likely candidates to represent RNA. Some of the trees previously identified as candidate structures are found to be "very" RNA like, while others are not, thereby refining the space of structures likely to be found as representing secondary RNA structure. Conclusion Search algorithms are available that mine nucleotide sequence databases. However, the number of motifs identified can be quite large, making a further search for similar motif computationally difficult. Much of the work in the bioinformatics arena is toward the development of better algorithms to address the computational problem. This work, on the other hand, uses mathematical descriptors to more clearly characterize the RNA motifs and thereby reduce the corresponding search space. These

  13. A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

    Directory of Open Access Journals (Sweden)

    Coupland George

    2005-08-01

    Full Text Available Abstract Background Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems. Results We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley. Conclusion To facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler ecotypes.

  14. Switch to raltegravir-based regimens and HIV DNA decrease in patients with suppressed HIV RNA

    Directory of Open Access Journals (Sweden)

    Claudia Bianco

    2014-11-01

    Full Text Available Introduction: Raltegravir intensification is associated with an increase in 2-LTR episomal HIV DNA= circles, indicating a persistent low-level replication, in some individuals in ART with suppressed HIV RNA. We aimed at monitoring residual plasma HIV RNA and cellular HIV DNA in virologically suppressed patients switching to a raltegravir-based regimen. Materials and Methods: Forty-six HIV-infected subjects on PI or NNRTI based-regimens, with plasma HIV RNA level 200 cells/µL for ≥12 months were enrolled. Thirty-four patients switched to raltegravir-based regimen (RASTA study group and 12 continued a PI or NNRTI based-regimen (control group. Ultrasensitive HIV residual viremia and total PBMC HIV DNA were assessed at baseline (W0, 24 (W24 and 48 (W48 weeks. HIV RNA levels were determined by an ultrasensitive test derived from a commercial real time PCR (limit of detection 5 copies/ml. A real time PCR was used to quantify HIV DNA copy numbers in PBMCs. Results: At W0, HIV DNA was detected in all patients while at W48 it was detectable in 82.3% of RASTA group vs 100% of controls (p=0.01. The difference between the average values of HIV DNA log10 copies/10°6 CD4 at W0 (median 3.11, IQR 2.70–3.45 and W48 (median 2.87, IQR 2.24–3.38 was statistically significant for RASTA group (p=0.035. Male gender (mean difference −0.37 log10 copies/10°6 PBMC, p=0.023 and previous PI based-ART (mean difference +0.39 log10 copies/10°6 PBMC, p=0.036 were predictive of HIV DNA level at W0. After adjusting for previous PI based-ART, male gender was the only variable independently associated with HIV DNA size at W0 (mean difference −0.326 log10 copies/10°6 PBMC, 95% CI −0.641, −0.011 p=0.043. Ultrasensitive HIV-1 RNA was detectable at W0 in 50% of RASTA group versus 66.7% of controls and at W48 in 32.4% versus 45.5%, respectively. No differences were found between HIV RNA levels at W0 and W48 within and between the two groups. Conclusions: Switching to

  15. Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery.

    Science.gov (United States)

    Xu, Yongjie; Li, Dandan; Cheng, Wei; Hu, Rong; Sang, Ye; Yin, Yibing; Ding, Shijia; Ju, Huangxian

    2016-09-14

    A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis. PMID:27566360

  16. Study design requirements for RNA sequencing-based breast cancer diagnostics.

    Science.gov (United States)

    Mer, Arvind Singh; Klevebring, Daniel; Grönberg, Henrik; Rantalainen, Mattias

    2016-01-01

    Sequencing-based molecular characterization of tumors provides information required for individualized cancer treatment. There are well-defined molecular subtypes of breast cancer that provide improved prognostication compared to routine biomarkers. However, molecular subtyping is not yet implemented in routine breast cancer care. Clinical translation is dependent on subtype prediction models providing high sensitivity and specificity. In this study we evaluate sample size and RNA-sequencing read requirements for breast cancer subtyping to facilitate rational design of translational studies. We applied subsampling to ascertain the effect of training sample size and the number of RNA sequencing reads on classification accuracy of molecular subtype and routine biomarker prediction models (unsupervised and supervised). Subtype classification accuracy improved with increasing sample size up to N = 750 (accuracy = 0.93), although with a modest improvement beyond N = 350 (accuracy = 0.92). Prediction of routine biomarkers achieved accuracy of 0.94 (ER) and 0.92 (Her2) at N = 200. Subtype classification improved with RNA-sequencing library size up to 5 million reads. Development of molecular subtyping models for cancer diagnostics requires well-designed studies. Sample size and the number of RNA sequencing reads directly influence accuracy of molecular subtyping. Results in this study provide key information for rational design of translational studies aiming to bring sequencing-based diagnostics to the clinic. PMID:26830453

  17. Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery.

    Science.gov (United States)

    Xu, Yongjie; Li, Dandan; Cheng, Wei; Hu, Rong; Sang, Ye; Yin, Yibing; Ding, Shijia; Ju, Huangxian

    2016-09-14

    A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis.

  18. Single-Point Mutation Detection in RNA Extracts using Gold Nanoparticles Modified with Hydrophobic Molecular Beacon-Like Structures

    Science.gov (United States)

    Latorre, Alfonso; Posch, Christian; Garcimartín, Yolanda; Ortiz-Urda, Susana; Somoza, Álvaro

    2015-01-01

    Gold nanoparticles functionalized with oligonucleotides that bear a cholesterol group are used as gene sensors. The hydrophobic molecule is buried inside the nanostructure but when the complementary RNA sequence is present the structure unfolds exposing the cholesterol group to the water. This rearrangement leads to the aggregation of the nanostructures. PMID:24496380

  19. Classification of Textures Using Filter Based Local Feature Extraction

    Directory of Open Access Journals (Sweden)

    Bocekci Veysel Gokhan

    2016-01-01

    Full Text Available In this work local features are used in feature extraction process in image processing for textures. The local binary pattern feature extraction method from textures are introduced. Filtering is also used during the feature extraction process for getting discriminative features. To show the effectiveness of the algorithm before the extraction process, three different noise are added to both train and test images. Wiener filter and median filter are used to remove the noise from images. We evaluate the performance of the method with Naïve Bayesian classifier. We conduct the comparative analysis on benchmark dataset with different filtering and size. Our experiments demonstrate that feature extraction process combine with filtering give promising results on noisy images.

  20. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    Science.gov (United States)

    Roh, Changhyun; Lee, Ho-Young; Kim, Sang-Eun; Jo, Sung-Kee

    2010-01-01

    Globally, approximately 170 million people (representing approximately 3% of the population worldwide), are infected with hepatitis C virus (HCV) and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs)-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B) on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinker™ glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL−1 to 1 ng mL−1 range with a detection limit of 1 ng mL−1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well. PMID:20517476

  1. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    Directory of Open Access Journals (Sweden)

    Shu Wu

    Full Text Available Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9 within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%; and could aid in species-level analyses, but with some limitations; 2 nearly-whole-length sequences and some partial regions (around V2, V4, and V9 of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%; 3 compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%; and 4 V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  2. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    Science.gov (United States)

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  3. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  4. Ionization and fragmentation of DNA-RNA bases: a density functional theory study

    International Nuclear Information System (INIS)

    Ionizing radiation (IR) cross human tissue, deposit energy and dissipate fragmenting molecules. The resulting fragments may be highlighted by mass spectrometry. Despite the amount of information obtained experimentally by the interpretation of the mass spectrum, experience alone cannot answer all the questions of the mechanism of fragmentation of DNA/RNA bases and a theoretical study is a complement to this information. A theoretical study allows us to know the weakest bonds in the molecule during ionization and thus may help to provide mechanisms of dissociation and produced fragments. The purpose of this work, using the DFT with the PBE functional, is to study the ionization and fragmentation mechanisms of DNA/RNA bases (Uracil, Cytosine, Adenine and Guanine) and to identify the cations corresponding to each peak in mass spectra. For all RNA bases, the retro Diels-Alder reaction (elimination of HNCO or NCO*) is a major route for dissociating, with the exception of adenine for which there is no atom oxygen in its structure. Loss of NH3 (NH2*) molecule is another common way to all bases that contain amine group. The possibility of the loss of hydrogen from the cations is also investigated, as well as the dissociation of dehydrogenated cations and protonated uracil. This work shows the interest of providing DFT calculation in the interpretation of mass spectra of DNA bases. (author)

  5. lncRNATargets: A platform for lncRNA target prediction based on nucleic acid thermodynamics.

    Science.gov (United States)

    Hu, Ruifeng; Sun, Xiaobo

    2016-08-01

    Many studies have supported that long noncoding RNAs (lncRNAs) perform various functions in various critical biological processes. Advanced experimental and computational technologies allow access to more information on lncRNAs. Determining the functions and action mechanisms of these RNAs on a large scale is urgently needed. We provided lncRNATargets, which is a web-based platform for lncRNA target prediction based on nucleic acid thermodynamics. The nearest-neighbor (NN) model was used to calculate binging-free energy. The main principle of NN model for nucleic acid assumes that identity and orientation of neighbor base pairs determine stability of a given base pair. lncRNATargets features the following options: setting of a specific temperature that allow use not only for human but also for other animals or plants; processing all lncRNAs in high throughput without RNA size limitation that is superior to any other existing tool; and web-based, user-friendly interface, and colored result displays that allow easy access for nonskilled computer operators and provide better understanding of results. This technique could provide accurate calculation on the binding-free energy of lncRNA-target dimers to predict if these structures are well targeted together. lncRNATargets provides high accuracy calculations, and this user-friendly program is available for free at http://www.herbbol.org:8001/lrt/ . PMID:27306075

  6. A simple and efficient parameter extraction procedure for physics based IGBT models

    OpenAIRE

    Rui Filipe Marques Chibante; Armando Luís Sousa Araújo; Adriano da Silva Carvalho

    2004-01-01

    Extraction of parameters for models of power semiconductors is a need for researchers working with development of power circuits. One of the drawbacks of physics based models is how to extract the numerous parameters to describe the model. Different approaches have been taken, most of them cumbersome to be solved.This paper presents a simple and accurate method of parameter extraction for physics based IGBT models. The procedure, based on an optimization algorithm (simulated annealing), is ea...

  7. Evidence for a base triple in the free HIV-1 TAR RNA

    OpenAIRE

    Huthoff, Hendrik; GIRARD, FREDERIC; Wijmenga, Sybren S.; Berkhout, Ben

    2004-01-01

    We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free T...

  8. Apriori and N-gram Based Chinese Text Feature Extraction Method

    Institute of Scientific and Technical Information of China (English)

    王晔; 黄上腾

    2004-01-01

    A feature extraction, which means extracting the representative words from a text, is an important issue in text mining field. This paper presented a new Apriori and N-gram based Chinese text feature extraction method, and analyzed its correctness and performance. Our method solves the question that the exist extraction methods cannot find the frequent words with arbitrary length in Chinese texts. The experimental results show this method is feasible.

  9. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens.

    Science.gov (United States)

    Li, R; Mock, R; Huang, Q; Abad, J; Hartung, J; Kinard, G

    2008-12-01

    A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

  10. Historical Feature Pattern Extraction Based Network Attack Situation Sensing Algorithm

    Directory of Open Access Journals (Sweden)

    Yong Zeng

    2014-01-01

    Full Text Available The situation sequence contains a series of complicated and multivariate random trends, which are very sudden, uncertain, and difficult to recognize and describe its principle by traditional algorithms. To solve the above questions, estimating parameters of super long situation sequence is essential, but very difficult, so this paper proposes a situation prediction method based on historical feature pattern extraction (HFPE. First, HFPE algorithm seeks similar indications from the history situation sequence recorded and weighs the link intensity between occurred indication and subsequent effect. Then it calculates the probability that a certain effect reappears according to the current indication and makes a prediction after weighting. Meanwhile, HFPE method gives an evolution algorithm to derive the prediction deviation from the views of pattern and accuracy. This algorithm can continuously promote the adaptability of HFPE through gradual fine-tuning. The method preserves the rules in sequence at its best, does not need data preprocessing, and can track and adapt to the variation of situation sequence continuously.

  11. A Logic-Based Approach to Relation Extraction from Texts

    Science.gov (United States)

    Horváth, Tamás; Paass, Gerhard; Reichartz, Frank; Wrobel, Stefan

    In recent years, text mining has moved far beyond the classical problem of text classification with an increased interest in more sophisticated processing of large text corpora, such as, for example, evaluations of complex queries. This and several other tasks are based on the essential step of relation extraction. This problem becomes a typical application of learning logic programs by considering the dependency trees of sentences as relational structures and examples of the target relation as ground atoms of a target predicate. In this way, each example is represented by a definite first-order Horn-clause. We show that an adaptation of Plotkin's least general generalization (LGG) operator can effectively be applied to such clauses and propose a simple and effective divide-and-conquer algorithm for listing a certain set of LGGs. We use these LGGs to generate binary features and compute the hypothesis by applying SVM to the feature vectors obtained. Empirical results on the ACE-2003 benchmark dataset indicate that the performance of our approach is comparable to state-of-the-art kernel methods.

  12. PDGI-BASED REGULAR SWEPT SURFACE EXTRACTION FROM POINT CLOUD

    Institute of Scientific and Technical Information of China (English)

    LI Jiangxiong; KE Yinglin; LI An; ZHU Weidong

    2006-01-01

    A principal direction Gaussian image (PDGI)-based algorithm is proposed to extract the regular swept surface from point cloud. Firstly, the PDGI of the regular swept surface is constructed from point cloud, then the bounding box of the Gaussian sphere is uniformly partitioned into a number of small cubes (3D grids) and the PDGI points on the Gaussian sphere are associated with the corresponding 3D grids. Secondly, cluster analysis technique is used to sort out a group of 3D grids containing more PDGI points among the 3D grids. By the connected-region growing algorithm, the congregation point or the great circle is detected from the 3D grids. Thus the translational direction is determined by the congregation point and the direction of the rotational axis is determined by the great circle. In addition, the positional point of the rotational axis is obtained by the intersection of all the projected normal lines of the rotational surface on the plane being perpendicular to the estimated direction of the rotational axis. Finally, a pattern search method is applied to optimize the translational direction and the rotational axis. Some experiments are used to illustrate the feasibility of the above algorithm.

  13. A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

    Directory of Open Access Journals (Sweden)

    M Heydarnezhadi

    2011-09-01

    Full Text Available Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

  14. A new anionic exchange stir bar sorptive extraction coating based on monolithic material for the extraction of inorganic anion.

    Science.gov (United States)

    Huang, Xiaojia; Lin, Jianbing; Yuan, Dongxing

    2010-07-23

    A novel anionic exchange stir bar sorptive extraction (SBSE) coating based on poly(2-(methacryloyloxy)ethyltrimethylammonium chloride-co-divinylbenzene) monolithic material for the extraction of inorganic anion was prepared. The effect of preparation conditions such as ratio of functional monomer to cross-linker, content of porogenic solvent on the extraction efficiencies were investigated in detailed. The monolithic material was characterized by elemental analysis, scanning electron microscopy and infrared spectroscopy. In order to investigate the extraction capacity of the new coating for inorganic anion, the new SBSE was combined with ionic chromatography with conductivity detection, Br-, NO3-, PO4(3-) and SO4(2-) were selected as detected solutes. Several extractive parameters, including pH value and ionic strength in sample matrix, desorption solvent, extraction and desorption time were optimized. The results showed that strongly ionic strength did not favor the extraction of anlaytes. Under the optimum experimental conditions, low detection limits (S/N=3) and quantification limits (S/N=10) of the proposed method for the target anions were achieved within the range of 0.92-2.62 and 3.03-9.25 microg/L, respectively. The method also showed good linearity, simplicity, practicality and low cost for the extraction inorganic anions. Finally, the proposed method was successfully used to detect the two different trademarks of commercial purified water with satisfactory recovery in the range of 70.0-92.6%. To the best of our knowledge, this is the first to use SBSE to enrich inorganic anions. PMID:20576270

  15. Polyphenolic extracts from cowpea (Vigna unguiculata) protect colonic myofibroblasts (CCD18Co cells) from lipopolysaccharide (LPS)-induced inflammation--modulation of microRNA 126.

    Science.gov (United States)

    Ojwang, Leonnard O; Banerjee, Nivedita; Noratto, Giuliana D; Angel-Morales, Gabriela; Hachibamba, Twambo; Awika, Joseph M; Mertens-Talcott, Susanne U

    2015-01-01

    Cowpea (Vigna unguiculata) is a drought tolerant crop with several agronomic advantages over other legumes. This study evaluated varieties from four major cowpea phenotypes (black, red, light brown and white) containing different phenolic profiles for their anti-inflammatory property on non-malignant colonic myofibroblasts (CCD18Co) cells challenged with an endotoxin (lipopolysaccharide, LPS). Intracellular reactive oxygen species (ROS) assay on the LPS-stimulated cells revealed antioxidative potential of black and red cowpea varieties. Real-time qRT-PCR analysis in LPS-stimulated cells revealed down-regulation of proinflammatory cytokines (IL-8, TNF-α, VCAM-1), transcription factor NF-κB and modulation of microRNA-126 (specific post-transcriptional regulator of VCAM-1) by cowpea polyphenolics. The ability of cowpea polyphenols to modulate miR-126 signaling and its target gene VCAM-1 were studied in LPS-stimulated endothelial cells transfected with a specific inhibitor of miR-126, and treated with 10 mg GAE/L black cowpea extract where the extract in part reversed the effect of the miR-126 inhibitor. This suggests that cowpea may exert their anti-inflammatory activities at least in part through induction of miR-126 that then down-regulate VCAM-1 mRNA and protein expressions. Overall, Cowpea therefore is promising as an anti-inflammatory dietary component. PMID:25300227

  16. An improved approach for flow-based cloud point extraction.

    Science.gov (United States)

    Frizzarin, Rejane M; Rocha, Fábio R P

    2014-04-11

    Novel strategies are proposed to circumvent the main drawbacks of flow-based cloud point extraction (CPE). The surfactant-rich phase (SRP) was directly retained into the optical path of the spectrophotometric cell, thus avoiding its dilution previously to the measurement and yielding higher sensitivity. Solenoid micro-pumps were exploited to improve mixing by the pulsed flow and also to modulate the flow-rate for retention and removal of the SRP, thus avoiding the elution step, often carried out with organic solvents. The heat released and the increase of the salt concentration provided by an on-line neutralization reaction were exploited to induce the cloud point without an external heating device. These innovations were demonstrated by the spectrophotometric determination of iron, yielding a linear response from 10 to 200 μg L(-1) with a coefficient of variation of 2.3% (n=7). Detection limit and sampling rate were estimated at 5 μg L(-1) (95% confidence level) and 26 samples per hour, respectively. The enrichment factor was 8.9 and the procedure consumed only 6 μg of TAN and 390 μg of Triton X-114 per determination. At the 95% confidence level, the results obtained for freshwater samples agreed with the reference procedure and those obtained for digests of bovine muscle, rice flour, brown bread and tort lobster agreed with the certified reference values. The proposed procedure thus shows advantages in relation to previously proposed approaches for flow-based CPE, being a fast and environmental friendly alternative for on-line separation and pre-concentration.

  17. Modified RNA extraction from field woody plants for the routine detection of PDV and PNRSV in cherry by RT-PCR%改良RNA提取法及樱桃PDV和PNRSV的RT-PCR检测

    Institute of Scientific and Technical Information of China (English)

    阮小凤; Jelkmann Wilhelm; 马锋旺

    2004-01-01

    介绍了一种从木本植物组织中获得高质量RNA的快速、简单和高效的核酸提取方法.该方法是基于核酸的二氧化硅捕获,避免了使用苯酚、氯仿等有机溶剂.利用该方法从樱桃组织中提取的总RNA用RT-PCR技术检测PDV,PNRSV均获得成功.从感病植株的一年生枝的叶片、韧皮部及芽组织中扩增出了预期的目的片段,即172和449 bp,而健康组织中无此扩增带.该法提取的总RNA用于RT-PCR技术检测,其敏感性至少与商业出售的Qiagen RNeasy提取试剂盒相当,但简单经济.%An efficient and effective procedure for the extraction of high-quality RNA from woody plants without the use of phenol,organic solvents,or alcohol precipitation is described,which is based on silica capture.The method described has been successfully used for the detection of PDV and PNRSV in cherry by RT-PCR assay using DNA primers for the viral coat protein region.The expected sizes of the amplified products were 172 and 449 bp.Samples from bark,leaves and buds were used.Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as the methods previously described using the commercially available Qiagen's RNeasy extraction kit.

  18. Microeukaryotic community and oxygen response in rice field soil revealed using a combined rRNA-gene and rRNA-based approach.

    Science.gov (United States)

    Murase, Jun; Takenouchi, Yuriko; Iwasaki, Kazufumi; Kimura, Makoto

    2014-01-01

    Irrigated rice field soil is subjected to frequent changes in oxygen status due to the water regime by agricultural management. In this study, the community response of microeukaryotes in rice field soil to the oxygen status was explored in a microcosm experiment under defined conditions. Water-saturated soil was incubated under a two-level factorial design of oxygen and organic enrichment with plant residue. The eukaryotic microbial community composition, which was either present or potentially active in the soils, was analyzed using denaturing gradient gel electrophoresis (DGGE) targeting the 18S rRNA gene or reverse-transcribed 18S rRNA. Oxygen availability was a primary factor shaping the microeukaryotic community in both DNA- and RNA-based analyses, revealing a shift within a week of incubation. Plant residue also affected the microeukaryotic community, which was more notable in the active community showing rRNA expression with time. Sequences of amplicons in DGGE bands indicated that protozoa (ciliates, flagellates, and amoebae) were the most prominent microeukaryotes in water-saturated rice field soil both in DNA- and RNA-based analyses. The use of a modified primer for soil protozoa suggested the functional importance of Heterolobosea amoeba in rice field soil, particularly in anoxic soil with organic enrichment.

  19. Biofunction-assisted DNA detection through RNase H-enhanced 3' processing of a premature tRNA probe in a wheat germ extract.

    Science.gov (United States)

    Ogawa, Atsushi; Tabuchi, Junichiro; Doi, Yasunori; Takamatsu, Masashi

    2016-08-01

    We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity. PMID:27289318

  20. Purification of form AI and AII DNA-dependent RNA polymerases from rat-liver nucleoli using low-ionic-strength extraction conditions.

    Science.gov (United States)

    Coupar, B E; Chesterton, C J

    1975-11-01

    Recent findings have confirmed the role of form A DNA-dependent polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method for the purification of the form AI and AII enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms AI and AII are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia coli RNA polymerase (Mr about 500,000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected.

  1. IsoLasso: A LASSO Regression Approach to RNA-Seq Based Transcriptome Assembly

    Science.gov (United States)

    Li, Wei; Feng, Jianxing; Jiang, Tao

    The new second generation sequencing technology revolutionizes many biology related research fields, and posts various computational biology challenges. One of them is transcriptome assembly based on RNA-Seq data, which aims at reconstructing all full-length mRNA transcripts simultaneously from millions of short reads. In this paper, we consider three objectives in transcriptome assembly: the maximization of prediction accuracy, minimization of interpretation, and maximization of completeness. The first objective, the maximization of prediction accuracy, requires that the estimated expression levels based on assembled transcripts should be as close as possible to the observed ones for every expressed region of the genome. The minimization of interpretation follows the parsimony principle to seek as few transcripts in the prediction as possible. The third objective, the maximization of completeness, requires that the maximum number of mapped reads (or "expressed segments" in gene models) be explained by (i.e., contained in) the predicted transcripts in the solution. Based on the above three objectives, we present IsoLasso, a new RNA-Seq based transcriptome assembly tool. IsoLasso is based on the well-known LASSO algorithm, a multivariate regression method designated to seek a balance between the maximization of prediction accuracy and the minimization of interpretation. By including some additional constraints in the quadratic program involved in LASSO, IsoLasso is able to make the set of assembled transcripts as complete as possible. Experiments on simulated and real RNA-Seq datasets show that IsoLasso achieves higher sensitivity and precision simultaneously than the state-of-art transcript assembly tools.

  2. Identification of threats using linguistics-based knowledge extraction.

    Energy Technology Data Exchange (ETDEWEB)

    Chew, Peter A.

    2008-09-01

    One of the challenges increasingly facing intelligence analysts, along with professionals in many other fields, is the vast amount of data which needs to be reviewed and converted into meaningful information, and ultimately into rational, wise decisions by policy makers. The advent of the world wide web (WWW) has magnified this challenge. A key hypothesis which has guided us is that threats come from ideas (or ideology), and ideas are almost always put into writing before the threats materialize. While in the past the 'writing' might have taken the form of pamphlets or books, today's medium of choice is the WWW, precisely because it is a decentralized, flexible, and low-cost method of reaching a wide audience. However, a factor which complicates matters for the analyst is that material published on the WWW may be in any of a large number of languages. In 'Identification of Threats Using Linguistics-Based Knowledge Extraction', we have sought to use Latent Semantic Analysis (LSA) and other similar text analysis techniques to map documents from the WWW, in whatever language they were originally written, to a common language-independent vector-based representation. This then opens up a number of possibilities. First, similar documents can be found across language boundaries. Secondly, a set of documents in multiple languages can be visualized in a graphical representation. These alone offer potentially useful tools and capabilities to the intelligence analyst whose knowledge of foreign languages may be limited. Finally, we can test the over-arching hypothesis--that ideology, and more specifically ideology which represents a threat, can be detected solely from the words which express the ideology--by using the vector-based representation of documents to predict additional features (such as the ideology) within a framework based on supervised learning. In this report, we present the results of a three-year project of the same name. We believe

  3. Modified TRIzol Method for RNA and DNA Co-extraction from Blood%改良TRIzol法同步提取血液RNA和DNA

    Institute of Scientific and Technical Information of China (English)

    秦娟娟; 路志勇; 焦章平; 朱晓君; 王颖希; 唐晖

    2013-01-01

    Objective To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent.Methods After the aqueous phase which contained total RNA was removed by traditional TRIzol method,the values of pH of the interphase phase and organic phase were adjusted.The DNA was precipitated with ethanol and purified with DNA IQTM system.The purified DNA was measured in quality and quantity.As the template,it was amplified and typed by PCR-STR.The data was compared with that extracted by traditional TRIzol method.Results The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing.Conclusion The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample.It could be used for individual identification and paternity testing to satisfy the need of forensic science.%目的 建立一种应用TRIzol试剂针对同一生物检材在提取总RNA的同时又能提取DNA的新方法. 方法 使用传统TRIzol法将含有总RNA的水相移除,调节中间层和有机相的pH值,乙醇沉淀DNA,运用DNA IQTM system试剂盒纯化DNA.检测DNA纯度和质量浓度,并以之为模板进行PCR-STR分型.与传统TRIzol法提取的基因组DNA进行比较. 结果 改良TRIzol法提取的基因组DNA较传统TRIzol法提取的基因组DNA纯度和质量浓度高,扩增后STR分型良好. 结论 改良TRIzol法可靠易行,能够实现同一样本同时提取RNA和DNA的目的,可以满足法庭科学个体识别和亲缘鉴定的要求.

  4. Circularity and self-cleavage as a strategy for the emergence of a chromosome in the RNA-based protocell

    OpenAIRE

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao

    2013-01-01

    Background It is now popularly accepted that an “RNA world” existed in early evolution. During division of RNA-based protocells, random distribution of individual genes (simultaneously as ribozymes) between offspring might have resulted in gene loss, especially when the number of gene types increased. Therefore, the emergence of a chromosome carrying linked genes was critical for the prosperity of the RNA world. However, there were quite a few immediate difficulties for this event to occur. F...

  5. An RNA-aptamer-based two-color CRISPR labeling system

    Science.gov (United States)

    Wang, Siyuan; Su, Jun-Han; Zhang, Feng; Zhuang, Xiaowei

    2016-01-01

    The spatial organization and dynamics of chromatin play important roles in essential biological functions. However, direct visualization of endogenous genomic loci in living cells has proven to be laborious until the recent development of CRISPR-Cas9-based chromatin labeling methods. These methods rely on the recognition of specific DNA sequences by CRISPR single-guide RNAs (sgRNAs) and fluorescent–protein-fused catalytically inactive Cas9 to label specific chromatin loci in cells. Previously, multicolor chromatin labeling has been achieved using orthogonal Cas9 proteins from different bacterial species fused to different fluorescent proteins. Here we report the development of an alternative two-color CRISPR labeling method using only the well-characterized Streptococcus pyogenes Cas9, by incorporating MS2 or PP7 RNA aptamers into the sgRNA. The MS2 or PP7 aptamers then recruit the corresponding MS2 or PP7 coat proteins fused with different fluorescent proteins to the target genomic loci. Here we demonstrate specific and orthogonal two-color labeling of repetitive sequences in living human cells using this method. By attaching the MS2 or PP7 aptamers to different locations on the sgRNA, we found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-background ratio of chromatin imaging. PMID:27229896

  6. The emergence of ribozymes synthesizing membrane components in RNA-based protocells.

    Science.gov (United States)

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Zhou, Ping; Hu, Jiming

    2010-03-01

    A significant problem of the origin of life is the emergence of cellular self-replication. In the context of the "RNA world", a crucial concern is how the RNA-based protocells could achieve the ability to produce their own membrane. Here we show, with the aid of a computer simulation, that for these protocells, there would be "immediately" a selection pressure for the emergence of a ribozyme synthesizing membrane components. The ribozyme would promote the enlargement of cellular space and favor the incoming (by permeation) of RNA's precursors, thus benefit the replication of inner RNA, including itself. Via growth and division, protocells containing the ribozyme would achieve superiority and spread in the system, and meanwhile the ribozyme would spread in the system. The present work is inspiring because it suggests that the transition from molecular self-replication to cellular self-replication might have occurred naturally (and necessarily) in the origin of life, leading to the emergence of Darwinian evolution at the cellular level. PMID:19961895

  7. Rank-Based miRNA Signatures for Early Cancer Detection

    Directory of Open Access Journals (Sweden)

    Mario Lauria

    2014-01-01

    Full Text Available We describe a new signature definition and analysis method to be used as biomarker for early cancer detection. Our new approach is based on the construction of a reference map of transcriptional signatures of both healthy and cancer affected individuals using circulating miRNA from a large number of subjects. Once such a map is available, the diagnosis for a new patient can be performed by observing the relative position on the map of his/her transcriptional signature. To demonstrate its efficacy for this specific application we report the results of the application of our method to published datasets of circulating miRNA, and we quantify its performance compared to current state-of-the-art methods. A number of additional features make this method an ideal candidate for large-scale use, for example, as a mass screening tool for early cancer detection or for at-home diagnostics. Specifically, our method is minimally invasive (because it works well with circulating miRNA, it is robust with respect to lab-to-lab protocol variability and batch effects (it requires that only the relative ranking of expression value of miRNA in a profile be accurate not their absolute values, and it is scalable to a large number of subjects. Finally we discuss the need for HPC capability in a widespread application of our or similar methods.

  8. Trends in evolution of 5S rRNA of deuterostomes: bases and homogeneous clusters

    Directory of Open Access Journals (Sweden)

    Sandra Maria Rodrigues Subacius

    2002-01-01

    Full Text Available Evolution of metazoan 5S rRNA sequences was analyzed through base composition and types, location and frequency of clustered bases. Characters from sequences of protostomes did not show regular trends as compared with paleontology dating or organism complexity. Trends of increasing G and C, stronger in G clusters, and decreasing A and U, were detected in deuterostomes, in parallel with evolution of complexity. The multifunctional domain 71-104 was highlighted among conserved stretches. Clusters of C were typical of helices. Those of G were longer, extending from helices into loops or related to bulges, which is suggestive of functional significance. Deuterostomian trends were installed early in the lineage and reached full development in aquatic organisms, not increasing further after reptiles. It can be suggested that ribosomal RNA structures participated in deuterostomian high regulatory complexity, either specifically or as part of the widespread processes of chromosomal regionalization.

  9. A grammar based methodology for structural motif finding in ncRNA database search.

    Science.gov (United States)

    Quest, Daniel; Tapprich, William; Ali, Hesham

    2007-01-01

    In recent years, sequence database searching has been conducted through local alignment heuristics, pattern-matching, and comparison of short statistically significant patterns. While these approaches have unlocked many clues as to sequence relationships, they are limited in that they do not provide context-sensitive searching capabilities (e.g. considering pseudoknots, protein binding positions, and complementary base pairs). Stochastic grammars (hidden Markov models HMMs and stochastic context-free grammars SCFG) do allow for flexibility in terms of local context, but the context comes at the cost of increased computational complexity. In this paper we introduce a new grammar based method for searching for RNA motifs that exist within a conserved RNA structure. Our method constrains computational complexity by using a chain of topology elements. Through the use of a case study we present the algorithmic approach and benchmark our approach against traditional methods.

  10. Extraction of MHD Signal Based on Wavelet Transform

    Institute of Scientific and Technical Information of China (English)

    赵晴初; 赵彤; 李旻; 黄胜华; 徐佩霞

    2002-01-01

    Mirnov signals mixed with interferences are a kind of non-stationary signal. It can not obtain satisfactory effects to extract MHD signals from mirnov signals by Fourier Transform. This paper suggests that the wavelet transform can be used to treat mirnov signals. Theoretical analysis and experimental result have indicated that using the time-frequency analysis characteristics of the wavelet transform to filter mirnov signals can remove effectively interferences and extract useful MHD signals.

  11. Bacterial Transcription Inhibitor of RNA Polymerase Holoenzyme Formation by Structure-Based Drug Design: From in Silico Screening to Validation.

    Science.gov (United States)

    Ma, Cong; Yang, Xiao; Lewis, Peter J

    2016-01-01

    Bacterial transcription is a proven target for antibacterial research. However, most of the known inhibitors targeting transcription are from natural extracts or are hits from screens where the binding site remains unidentified. Using an RNA polymerase holoenzyme homology structure from the model Gram-positive organism Bacillus subtilis, we created a pharmacophore model and used it for in silico screening of a publicly available library for compounds able to inhibit holoenzyme formation. The hits demonstrated specific affinity to bacterial RNA polymerase and excellent activity using in vitro assays and showed no binding to the equivalent structure from human RNA polymerase II. The target specificity in live cells and antibacterial activity was demonstrated in microscopy and growth inhibition experiments. This is the first example of targeted inhibitor development for a bacterial RNA polymerase, outlining a complete discovery process from virtual screening to biochemical validation. This approach could serve as an appropriate platform for the future identification of inhibitors of bacterial transcription. PMID:27622946

  12. Method for total RNA rapid extraction from mature tissues of fieldgrown apple%田间苹果成熟组织RNA高效提取方法

    Institute of Scientific and Technical Information of China (English)

    焦朝霞; 邵建柱; 孙建设

    2011-01-01

    The mature leaves of apple trees are rich in polyhexose, polyphenols and other secondary metabolism compounds which prevent RNA extraction. The genomic BNA samples of mature leaves and phloem of field-grown apple in different seasons were isolated by the improved method. Total RNA of high purity, higher content and good integrity could be obtained within about 2 hours by this method and could be well used for RT-PCR. The repetitious experiments indicated that this method was applicable to rapid isolation of RNA from apple trees for simple operation, rapidness, greatly time saving and reduction of using poisonous chemicals. It can be used to detect the viruses from a great amount of samples and can be widely used for research on viruses from fruit trees of various species in different seasons.%针对田间苹果成熟组织富含多酚、多糖和大量次生代谢产物的特点,建立了适合田间苹果不同组织的高效RNA提取方法,以不同季节田间生长的苹果叶片和皮层为材料,用此方法均能在2 h左右完成超过10个样品的总RNA提取,质量高、完整性好的总RNA,可用于田间苹果病毒RT-PCR检测,获得良好效果.经反复大量试验证实,该方法适合田间苹果不同组织的RNA快速提取,操作简便、快捷、高效,且使其不受季节限制,有效地提高了田间果树病毒检测以及其他分子生物学研究的效率.

  13. Growth Inhibition of Head and Neck Squamous Cell Carcinoma Cells by sgRNA Targeting the Cyclin D1 mRNA Based on TRUE Gene Silencing

    OpenAIRE

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any...

  14. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers.

    Science.gov (United States)

    Jolly, Pawan; Estrela, Pedro; Ladomery, Michael

    2016-06-30

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  15. Evolutionary patterns of RNA-based duplication in non-mammalian chordates.

    Directory of Open Access Journals (Sweden)

    Ming Chen

    Full Text Available The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.

  16. DIANA-TarBase and DIANA Suite Tools: Studying Experimentally Supported microRNA Targets.

    Science.gov (United States)

    Paraskevopoulou, Maria D; Vlachos, Ioannis S; Hatzigeorgiou, Artemis G

    2016-01-01

    microRNAs (miRNAs) are short non-coding RNAs (∼22 nts) present in animals, plants, and viruses. They are considered central post-transcriptional regulators of gene expression and are key components in a great number of physiological and pathological conditions. The accurate characterization of their targets is considered essential to a series of applications and basic or applied research settings. DIANA-TarBase (http://www.microrna.gr/tarbase) was initially launched in 2006. It is a reference repository indexing experimentally derived miRNA-gene interactions in different cell types, tissues, and conditions across numerous species. This unit focuses on the study of experimentally supported miRNA-gene interactions, as well as their functional interpretation through the use of available tools in the DIANA suite (http://www.microrna.gr). The proposed use-case scenarios are presented in protocols, describing how to utilize the DIANA-TarBase database and DIANA-microT-CDS server and perform miRNA-targeted pathway analysis with DIANA-miRPath-v3. All analyses are directly invoked or initiated from DIANA-TarBase. © 2016 by John Wiley & Sons, Inc. PMID:27603020

  17. Knowledge based system for runtime controlling of multiscale model of ion-exchange solvent extraction

    Science.gov (United States)

    Macioł, Piotr; Gotfryd, Leszek; Macioł, Andrzej

    2012-09-01

    The hereby paper concerns the issue of solution of runtime controlling of multiscale model of ion-exchange solvent extraction. It is based on cooperation of a framework applying Agile Multiscale Modeling Methodology (AM3), and the REBIT Knowledge Based System. Ion-exchange solvent extraction has been shortly introduced. Design assumptions of AM3 and theoretical basis of REBIT have been described. Designed workflows and rules for simple laminar/ turbulent flow and extraction processes have been shown.

  18. miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods.

    Science.gov (United States)

    Nagy, Zsófia Brigitta; Wichmann, Barnabás; Kalmár, Alexandra; Barták, Barbara Kinga; Tulassay, Zsolt; Molnár, Béla

    2016-07-01

    MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable.

  19. A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results

    DEFF Research Database (Denmark)

    van den Berge, M; Carracedo, A; Gomes, I;

    2014-01-01

    that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference...... of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains......The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions...

  20. Linking Α to Ω: diverse and dynamic RNA-based mechanisms to regulate gene expression by 5'-to-3' communication.

    Science.gov (United States)

    Filbin, Megan E; Kieft, Jeffrey S

    2016-01-01

    Communication between the 5' and 3' ends of a eukaryotic messenger RNA (mRNA) or viral genomic RNA is a ubiquitous and important strategy used to regulate gene expression. Although the canonical interaction between initiation factor proteins at the 5' end of an mRNA and proteins bound to the polyadenylate tail at the 3' end is well known, in fact there are many other strategies used in diverse ways. These strategies can involve "non-canonical" proteins, RNA structures, and direct RNA-RNA base-pairing between distal elements to achieve 5'-to-3' communication. Likewise, the communication induced by these interactions influences a variety of processes linked to the use and fate of the RNA that contains them. Recent studies are revealing how dynamic these interactions are, possibly changing in response to cellular conditions or to link various phases of the mRNA's life, from translation to decay. Thus, 5'-to-3' communication is about more than just making a closed circle; the RNA elements and associated proteins are key players in controlling gene expression at the post-transcriptional level. PMID:27610229

  1. Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins Efeito de polietilenoglicol e polivinilpirrolidona na extração e hibridização de rRNA bacteriano de células expostas a taninos

    OpenAIRE

    Pedro Braga Arcuri; Michael Larry Thonney; Peter Schofield; Alice Nelson Pell

    2003-01-01

    In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/...

  2. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    Directory of Open Access Journals (Sweden)

    Changhyun Roh

    2010-04-01

    Full Text Available Changhyun Roh1, Ho-Young Lee2, Sang-Eun Kim2, Sung-Kee Jo11Radiation Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Sinjeong-dong, Jeongeup, Jeonbuk, South Korea; 2Department of Nuclear Medicine, College of Medicine, Seoul National University, South KoreaAbstract: Globally, approximately 170 million people (representing approximately 3% of the population worldwide, are infected with hepatitis C virus (HCV and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinkerTM glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL-1 to 1 ng mL-1 range with a detection limit of 1 ng mL-1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well.Keywords: hepatitis C virus, viral protein, RNA oligonucleotide, quantum dots, biochip

  3. mRNA-based vaccines synergize with radiation therapy to eradicate established tumors

    International Nuclear Information System (INIS)

    The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics. The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells. In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors. Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies. The online version of this article (doi:10.1186/1748-717X-9-180) contains supplementary material, which is available to authorized users

  4. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    Energy Technology Data Exchange (ETDEWEB)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Baelum, Jacob; Tas, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Phillip; Prieme, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  5. Effects of Gingko biloba leaf extract on learning, memory, and hippocampal amyloid precursor protein mRNA expressions in diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Xiaofan Zhang; Bo Liang; Zhifeng Liang; Jun Lin

    2008-01-01

    BACKGROUND: The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein (APP) mRNA overexpression. Studies have shown that Gingko biloba leaf extract (Egb) is effective in promoting functional recovery of the brain after traumatic injury. Egb is also effective in improving central nervous system plasticity and learning and memory functions of the elderly.OBJECTIVE: To study the effects of Egb on learning and memory, as well as hippocampal APP mRNA expression in the brains of diabetic rats, using Morris water maze behavioral testing and reverse transcription polymerase chain reaction (RT-PCR), respectively.DESIGN: Complete random design, controlled experimental study.SETTING: Department of Pharmacology, Pharmaceutical School, Guangxi Medical University.MATERIALS: A total of 70 male Wistar rats (180-220 g), 8 weeks old and specific pathogen free, were used for this study. GbE (containing 24.8% flavone glycosides and 6.2% diterpene lactone) was purchased from Guilin Sitejia Natural Plants Pharmaceutical Factory (Guangxi Province, Lot NO. 200405). Streptozotocin was purchased from Sigma (USA). Protamine zinc insulin injection was purchased from WANBANG Biochemical Pharmaceutical Co., Ltd. (Xuzhou Jiangsu, China).METHODS: The experiment was performed in the Experimental Center of Guangxi Medical University from March to October 2005. ① Experimental intervention: 70 rats were divided randomly into normal control group, diabetic model group (DM group), diabetic model +10 μg/kg insulin group (DM + Ins group), diabetic model + 100 mg/kg ginkgo leaf extract group (DM + Egb high-dose group), and diabetic model + 50 mg/kg ginkgo leaf extract group (DM + Egb low-dose group); there were 14 rats in each group. Rats with an intraperitoneal (I.p.) injection of citrate buffer solution (pH 4.4) served as the control group. To establish the diabetes model, rats were treated with I.p. Injection of 55 mg/kg streptozotocin. Insulin (10 U/kg) was

  6. Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture.

    Science.gov (United States)

    Miassod, R; Cecchini, J P

    1979-04-26

    Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.

  7. Surface Electromyography Feature Extraction Based on Wavelet Transform

    Directory of Open Access Journals (Sweden)

    Farzaneh Akhavan Mahdavi

    2012-12-01

    Full Text Available Considering the vast variety of EMG signal applications such as rehabilitation of people suffering from some mobility limitations, scientists have done much research on EMG control system. In this regard, feature extraction of EMG signal has been highly valued as a significant technique to extract the desired information of EMG signal and remove unnecessary parts. In this study, Wavelet Transform (WT has been applied as the main technique to extract Surface EMG (SEMG features because WT is consistent with the nature of EMG as a nonstationary signal. Furthermore, two evaluation criteria, namely, RES index (the ratio of a Euclidean distance to a standard deviation and scatter plot are recruited to investigate the efficiency of wavelet feature extraction. The results illustrated an improvement in class separability of hand movements in feature space. Accordingly, it has been shown that only the SEMG features extracted from first and second level of WT decomposition by second order of Daubechies family (db2 yielded the best class separability.

  8. Comparison of RNA Extraction Method from Tomato Fruit%番茄果实RNA提取方法比较

    Institute of Scientific and Technical Information of China (English)

    马敬; 纪鸿飞; 李培

    2011-01-01

    从富含多糖、多酚的番茄果实中提取高质量的总RNA,对进行下一步分子生物学研究至关重要.采用CTAB-LiCl法和Trizol法分别对番茄果实的总RNA进行提取和分析.结果表明,CTAB-LiCl法提取的总RNA产量低,而且有基因组DNA的污染:Trizol方法提取的RNA产量相对较高,很少有基因组DNA污染.

  9. 辣椒组织总RNA提取方法研究%Study on RNA Extraction from Hot Pepper Tissues

    Institute of Scientific and Technical Information of China (English)

    李燕凌; 李大志

    2007-01-01

    从RNA的完整性、纯度和产量等方面比较了3种RNA提取试剂对辣椒不同组织部位RNA的提取效果.结果表明,RNAplant能从成熟组织中获得较高质量和产量的RNA,用该方法提取的辣椒老化组织RNA经RT-PCR能成功进行内参检测.

  10. Electrochemical based detection of microRNA, mir21 in breast cancer cells.

    Science.gov (United States)

    Kilic, Tugba; Topkaya, Seda Nur; Ozkan Ariksoysal, Dilsat; Ozsoz, Mehmet; Ballar, Petek; Erac, Yasemin; Gozen, Oguz

    2012-01-01

    In this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3' end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin-avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (α-NAP, the product). α-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from α-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6 pmol for cell lysates. PMID:22776181

  11. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: lxuhelen@163.com [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: liuzhiguo@fmmu.edu.cn [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  12. Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

    Science.gov (United States)

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne

    2014-06-01

    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

  13. Cleavable Molecular Beacon for Hg(2+) Detection Based on Phosphorothioate RNA Modifications.

    Science.gov (United States)

    Huang, Po-Jung Jimmy; Wang, Feng; Liu, Juewen

    2015-07-01

    Mercury is a highly toxic heavy metal, and detection of Hg(2+) by biosensors has attracted extensive research interest in the past decade. In particular, a number of DNA-based sensing strategies have been developed. Well-known examples include thymine-Hg(2+) interactions and Hg(2+)-activated DNAzymes. However, these mechanisms are highly dependent on buffer conditions or require hybridization with another DNA strand. Herein, we report a new mechanism based on Hg(2+)-induced cleavage of phosphorothioate (PS) modified RNA. Among the various metal ions tested, Hg(2+) induced the most significant cleavage (∼16%), while other metals cleaved less than 2% of the same substrate. The uncleaved substrate undergoes desulfurization in the presence of Hg(2+). This cleavage reaction yields a similar amount of product from pH 3.5 to 7 and in the temperature range between 20 and 90 °C. Various PS RNA junctions can be cleaved with a similar efficiency, but PS DNA junctions cannot be cleaved. A molecular beacon containing three PS RNA modifications is designed, detecting Hg(2+) down to 1.7 nM with excellent selectivity. This sensor can also detect Hg(2+) in the Lake Ontario water sample, although its response is significantly masked by fish tissues. PMID:26060876

  14. Ontology-based Knowledge Extraction from Hidden Web

    Institute of Scientific and Technical Information of China (English)

    SONG Hui; MA Fan-yuan; LIU Xiao-qiang

    2004-01-01

    Hidden Web provides great amount of domain-specific data for constructing knowledge services. Most previous knowledge extraction researches ignore the valuable data hidden in Web database, and related works do not refer how to make extracted information available for knowledge system. This paper describes a novel approach to build a domain-specific knowledge service with the data retrieved from Hidden Web. Ontology serves to model the domain knowledge. Queries forms of different Web sites are translated into machine-understandable format, defined knowledge concepts, so that they can be accessed automatically. Also knowledge data are extracted from Web pages and organized in ontology format knowledge. The experiment proves the algorithm achieves high accuracy and the system facilitates constructing knowledge services greatly.

  15. Social Network Extraction and Analysis Based on Multimodal Dyadic Interaction

    Directory of Open Access Journals (Sweden)

    Bogdan Raducanu

    2012-02-01

    Full Text Available Social interactions are a very important component in people’s lives. Social network analysis has become a common technique used to model and quantify the properties of social interactions. In this paper, we propose an integrated framework to explore the characteristics of a social network extracted from multimodal dyadic interactions. For our study, we used a set of videos belonging to New York Times’ Blogging Heads opinion blog. The Social Network is represented as an oriented graph, whose directed links are determined by the Influence Model. The links’ weights are a measure of the “influence” a person has over the other. The states of the Influence Model encode automatically extracted audio/visual features from our videos using state-of-the art algorithms. Our results are reported in terms of accuracy of audio/visual data fusion for speaker segmentation and centrality measures used to characterize the extracted social network.

  16. A Karnaugh-Map based fingerprint minutiae extraction method

    Directory of Open Access Journals (Sweden)

    Sunil Kumar Singla

    2010-07-01

    Full Text Available Fingerprint is one of the most promising method among all the biometric techniques and has been used for thepersonal authentication for a long time because of its wide acceptance and reliability. Features (Minutiae are extracted fromthe fingerprint in question and are compared with the features already stored in the database for authentication. Crossingnumber (CN is the most commonly used minutiae extraction method for fingerprints. In this paper, a new Karnaugh-Mapbased fingerprint minutiae extraction method has been proposed and discussed. In the proposed algorithm the 8 neighborsof a pixel in a 33 window are arranged as 8 bits of a byte and corresponding hexadecimal (hex value is calculated. Thesehex values are simplified using standard Karnaugh-Map (K-map technique to obtain the minimized logical expression.Experiments conducted on the FVC2002/Db1_a database reveals that the developed method is better than the crossingnumber (CN method.

  17. Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves.

    Science.gov (United States)

    Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang

    2016-01-15

    A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions.

  18. An RNA secondary structure prediction method based on minimum and suboptimal free energy structures.

    Science.gov (United States)

    Fu, Haoyue; Yang, Lianping; Zhang, Xiangde

    2015-09-01

    The function of an RNA-molecule is mainly determined by its tertiary structures. And its secondary structure is an important determinant of its tertiary structure. The comparative methods usually give better results than the single-sequence methods. Based on minimum and suboptimal free energy structures, the paper presents a novel method for predicting conserved secondary structure of a group of related RNAs. In the method, the information from the known RNA structures is used as training data in a SVM (Support Vector Machine) classifier. Our method has been tested on the benchmark dataset given by Puton et al. The results show that the average sensitivity of our method is higher than that of other comparative methods such as CentroidAlifold, MXScrana, RNAalifold, and TurboFold. PMID:26100179

  19. Analysis of energy-based algorithms for RNA secondary structure prediction

    Directory of Open Access Journals (Sweden)

    Hajiaghayi Monir

    2012-02-01

    Full Text Available Abstract Background RNA molecules play critical roles in the cells of organisms, including roles in gene regulation, catalysis, and synthesis of proteins. Since RNA function depends in large part on its folded structures, much effort has been invested in developing accurate methods for prediction of RNA secondary structure from the base sequence. Minimum free energy (MFE predictions are widely used, based on nearest neighbor thermodynamic parameters of Mathews, Turner et al. or those of Andronescu et al. Some recently proposed alternatives that leverage partition function calculations find the structure with maximum expected accuracy (MEA or pseudo-expected accuracy (pseudo-MEA methods. Advances in prediction methods are typically benchmarked using sensitivity, positive predictive value and their harmonic mean, namely F-measure, on datasets of known reference structures. Since such benchmarks document progress in improving accuracy of computational prediction methods, it is important to understand how measures of accuracy vary as a function of the reference datasets and whether advances in algorithms or thermodynamic parameters yield statistically significant improvements. Our work advances such understanding for the MFE and (pseudo-MEA-based methods, with respect to the latest datasets and energy parameters. Results We present three main findings. First, using the bootstrap percentile method, we show that the average F-measure accuracy of the MFE and (pseudo-MEA-based algorithms, as measured on our largest datasets with over 2000 RNAs from diverse families, is a reliable estimate (within a 2% range with high confidence of the accuracy of a population of RNA molecules represented by this set. However, average accuracy on smaller classes of RNAs such as a class of 89 Group I introns used previously in benchmarking algorithm accuracy is not reliable enough to draw meaningful conclusions about the relative merits of the MFE and MEA-based algorithms

  20. A crack extraction algorithm based on improved median filter and Hessian matrix

    Science.gov (United States)

    Zhao, Yafeng; Zhao, Qiancheng; He, Yongbiao; Lu, Guofeng

    2016-01-01

    Aiming at the problems of existing crack extraction algorithms which are difficult to achieve fast and accurate crack extraction of image, an algorithm of crack detection based on Median Filter and Hessian Matrix is proposed. Firstly, median filter of crack gray image in 4 directions, Level, 45 degree, vertical and -45 degree, is conducted, by which noises are removed and roughly extracted crack is obtained. Then according to the Hessian matrix feature of extracting image linear feature, convolution of Differential operation of the Hessian matrix is adopted, and crack is further extracted through eigenvalues response and changing standard deviation of Gaussian function. The proposed algorithm validity is verified by comparison with other crack extraction algorithm. The results show that this algorithm has obvious accuracy rate in crack extraction.

  1. Level Sets and Voronoi based Feature Extraction from any Imagery

    DEFF Research Database (Denmark)

    Sharma, O.; Anton, François; Mioc, Darka

    2012-01-01

    imagery, and 2D/3D acoustic images (from hydrographic surveys). The application involving satellite imagery shown in this paper is coastline detection, but the methodology can be easily applied to feature extraction on any king of imagery. A prototype application that is developed as part of this research...

  2. Extraction of alkaloids for NMR-based profiling

    DEFF Research Database (Denmark)

    Yilmaz, Ali; Nyberg, Nils; Jaroszewski, Jerzy W.

    2012-01-01

    of the well-known cinchona alkaloids quinine, cinchonine and cinchonidine without any apparent clustering. Signals from analogues were detected but not in substantial amounts. The main variation was related to the absolute amounts of extracted alkaloids, which was attributed to the evolution of the Cinchona...

  3. Coastline Extraction from Aerial Images Based on Edge Detection

    Science.gov (United States)

    Paravolidakis, V.; Moirogiorgou, K.; Ragia, L.; Zervakis, M.; Synolakis, C.

    2016-06-01

    Nowadays coastline extraction and tracking of its changes become of high importance because of the climate change, global warming and rapid growth of human population. Coastal areas play a significant role for the economy of the entire region. In this paper we propose a new methodology for automatic extraction of the coastline using aerial images. A combination of a four step algorithm is used to extract the coastline in a robust and generalizable way. First, noise distortion is reduced in order to ameliorate the input data for the next processing steps. Then, the image is segmented into two regions, land and sea, through the application of a local threshold to create the binary image. The result is further processed by morphological operators with the aim that small objects are being eliminated and only the objects of interest are preserved. Finally, we perform edge detection and active contours fitting in order to extract and model the coastline. These algorithmic steps are illustrated through examples, which demonstrate the efficacy of the proposed methodology.

  4. Model Based Analysis of Face Images for Facial Feature Extraction

    Science.gov (United States)

    Riaz, Zahid; Mayer, Christoph; Beetz, Michael; Radig, Bernd

    This paper describes a comprehensive approach to extract a common feature set from the image sequences. We use simple features which are easily extracted from a 3D wireframe model and efficiently used for different applications on a benchmark database. Features verstality is experimented on facial expressions recognition, face reognition and gender classification. We experiment different combinations of the features and find reasonable results with a combined features approach which contain structural, textural and temporal variations. The idea follows in fitting a model to human face images and extracting shape and texture information. We parametrize these extracted information from the image sequences using active appearance model (AAM) approach. We further compute temporal parameters using optical flow to consider local feature variations. Finally we combine these parameters to form a feature vector for all the images in our database. These features are then experimented with binary decision tree (BDT) and Bayesian Network (BN) for classification. We evaluated our results on image sequences of Cohn Kanade Facial Expression Database (CKFED). The proposed system produced very promising recognition rates for our applications with same set of features and classifiers. The system is also realtime capable and automatic.

  5. Apect-Based Opinion Extraction From Customer Reviews

    Directory of Open Access Journals (Sweden)

    Amani K Samha

    2014-04-01

    Full Text Available Text is the main method of communicating informatio n in the digital age. Messages, blogs, news articles, reviews, and opinionated information abounds on the Internet. People commonly purchase products online and post their opinions ab out purchased items. This feedback is displayed publicly to assist others with their purc hasing decisions, creating the need for a mechanism with which to extract and summarize usefu l information for enhancing the decision- making process. Our contribution is to improve the accuracy of extraction by combining different techniques from three major areas, namedD ata Mining, Natural Language Processing techniques and Ontologies. The proposed framework sequentially mines product’s aspects and users’ opinions, groups representative aspects by s imilarity, and generates an output summary. This paper focuses on the task of extracting produc t aspects and users’ opinions by extracting all possible aspects and opinions from reviews usin g natural language, ontology, and frequent “tag”sets. The proposed framework, when compared w ith an existing baseline model, yielded promising results.

  6. Rule set transferability for object-based feature extraction

    NARCIS (Netherlands)

    Anders, N.S.; Seijmonsbergen, Arie C.; Bouten, Willem

    2015-01-01

    Cirques are complex landforms resulting from glacial erosion and can be used to estimate Equilibrium Line Altitudes and infer climate history. Automated extraction of cirques may help research on glacial geomorphology and climate change. Our objective was to test the transferability of an object-

  7. Background Knowledge in Learning-Based Relation Extraction

    Science.gov (United States)

    Do, Quang Xuan

    2012-01-01

    In this thesis, we study the importance of background knowledge in relation extraction systems. We not only demonstrate the benefits of leveraging background knowledge to improve the systems' performance but also propose a principled framework that allows one to effectively incorporate knowledge into statistical machine learning models for…

  8. A novel technique for extracting clouds base height using ground based imaging

    Directory of Open Access Journals (Sweden)

    E. Hirsch

    2011-01-01

    Full Text Available The height of a cloud in the atmospheric column is a key parameter in its characterization. Several remote sensing techniques (passive and active, either ground-based or on space-borne platforms and in-situ measurements are routinely used in order to estimate top and base heights of clouds. In this article we present a novel method that combines thermal imaging from the ground and sounded wind profile in order to derive the cloud base height. This method is independent of cloud types, making it efficient for both low boundary layer and high clouds. In addition, using thermal imaging ensures extraction of clouds' features during daytime as well as at nighttime. The proposed technique was validated by comparison to active sounding by ceilometers (which is a standard ground based method, to lifted condensation level (LCL calculations, and to MODIS products obtained from space. As all passive remote sensing techniques, the proposed method extracts only the height of the lowest cloud layer, thus upper cloud layers are not detected. Nevertheless, the information derived from this method can be complementary to space-borne cloud top measurements when deep-convective clouds are present. Unlike techniques such as LCL, this method is not limited to boundary layer clouds, and can extract the cloud base height at any level, as long as sufficient thermal contrast exists between the radiative temperatures of the cloud and its surrounding air parcel. Another advantage of the proposed method is its simplicity and modest power needs, making it particularly suitable for field measurements and deployment at remote locations. Our method can be further simplified for use with visible CCD or CMOS camera (although nighttime clouds will not be observed.

  9. Improved extraction of fluoroquinolones with recyclable ionic-liquid-based aqueous biphasic systems†

    Science.gov (United States)

    Almeida, Hugo F. D.; Freire, Mara G.; Marrucho, Isabel M.

    2016-01-01

    In the past few years, the improvement of advanced analytical tools allowed to confirm the presence of trace amounts of metabolized and unchanged active pharmaceutical ingredients (APIs) in wastewater treatment plants (WWTPs) as well as in freshwater surfaces. It is known that the continuous contact with APIs, even at very low concentrations (ng L−1–μg L−1), leads to serious human health problems. In this context, this work shows the feasibility of using ionic-liquid-based aqueous biphasic systems (IL-based ABS) in the extraction of quinolones present in aqueous media. In particular, ABS composed of imidazolium- and phosphonium-based ILs and aluminium-based salts (already used in water treatment plants) were evaluated in one-step extractions of six fluoroquinolones (FQs), namely ciprofloxacin, enrofloxacin, moxifloxacin, norfloxacin, ofloxacin and sarafloxacin, and extraction efficiencies up to 98% were obtained. Despite the large interest devoted to IL-based ABS as extractive systems of outstanding performance, their recyclability/reusability has seldomly been studied. An efficient extraction/cleaning process of the IL-rich phase is here proposed by FQs induced precipitation. The recycling of the IL and its further reuse without losses in the ABS extractive performance for FQs were established, as confirmed by the four consecutive removal/extraction cycles evaluated. This novel recycling strategy supports IL-based ABS as sustainable and cost-efficient extraction platforms.

  10. Growth and nitrogen metabolism of sea bass fed graded levels of nucleic acid nitrogen from yeast or RNA extract as partial substitute for protein nitrogen from fish meal

    Directory of Open Access Journals (Sweden)

    S. Kaushik

    2010-01-01

    Full Text Available Some studies carried out in mammalian models have shown de novo synthesis and salvage of nucleotides to be a costly metabolic process and a dietary supplementation with nucleic acids (NA or nucleotides has been suggested to result in a protein sparing action (Sanderson and He, 1994. On the other hand, high levels of dietary NA could have toxic effects and lead to disturbance in protein, lipid and carbohydrate metabolism in monogastric animals lacking uricase activity, an enzyme involved in NA degradation (Clifford and Story, 1976. So far, there is no clear indication of such effects in fish fed nucleic acid-enriched diets (Tacon and Cooke, 1980; Rumsey et al., 1992; Fournier et al., 2002. The aim of this experiment was to investigate growth response and N metabolism in juvenile sea bass (D. labrax fed diets supplying graded levels of nucleic acid N from dry brewer's yeast or RNA extract as partial substitutes for protein nitrogen provided by fish meal.

  11. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-04-01

    Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.

  12. Feature Extraction with Ordered Mean Values for Content Based Image Classification

    Directory of Open Access Journals (Sweden)

    Sudeep Thepade

    2014-01-01

    Full Text Available Categorization of images into meaningful classes by efficient extraction of feature vectors from image datasets has been dependent on feature selection techniques. Traditionally, feature vector extraction has been carried out using different methods of image binarization done with selection of global, local, or mean threshold. This paper has proposed a novel technique for feature extraction based on ordered mean values. The proposed technique was combined with feature extraction using discrete sine transform (DST for better classification results using multitechnique fusion. The novel methodology was compared to the traditional techniques used for feature extraction for content based image classification. Three benchmark datasets, namely, Wang dataset, Oliva and Torralba (OT-Scene dataset, and Caltech dataset, were used for evaluation purpose. Performance measure after evaluation has evidently revealed the superiority of the proposed fusion technique with ordered mean values and discrete sine transform over the popular approaches of single view feature extraction methodologies for classification.

  13. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    Science.gov (United States)

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  14. Application of ionic liquid-based microwave-assisted extraction of flavonoids from Scutellaria baicalensis Georgi.

    Science.gov (United States)

    Zhang, Qin; Zhao, San-Hu; Chen, Jue; Zhang, Li-Wei

    2015-10-01

    In the present work, a rapid ionic liquid-based microwave-assisted extraction (ILMAE) method was successfully applied to simultaneous extraction of baicalin, wogonoside, baicalein and wogonin from Scutellaria baicalensis Georgi. A series of 1-alkyl-3-methylirnidazolium ionic liquids with different anions and cations were assessed for extraction efficiency, and 1-octyl-3-methylimidazolium bromide was selected as the optimal solvent. In addition, the parameters of ILMAE procedure for the four flavonoids were optimized, and the optimal ILMAE method was validated in the linearity, stability, precision and recovery. Meanwhile, the microstructures of S. baicalensis powders were observed before and after extraction with the help of a scanning electron microscope (SEM) in order to explore the extraction mechanism, and the activity of the crude enzyme solution from S. baicalensis was determined through the hydrolysis of baicalin. Finally, the extraction yields and extraction time of WaterHRE, WaterMAE, ILHRE and Chp were 5.18% (30min), 8.77% (90s), 16.94% (30min) and 18.58% (3h), respectively. The results indicated that compared with the conventional extraction approaches, ILMAE possessed great advantages in extracting flavonoids, such as the highest extraction yield (22.28%), the shortest extraction time (90s), etc.

  15. Ionic Liquid-Based Microwave-Assisted Extraction of Flavonoids from Bauhinia championii (Benth. Benth.

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2012-12-01

    Full Text Available An ionic liquids (IL-based microwave-assisted approach for extraction and determination of flavonoids from Bauhinia championii (Benth. Benth. was proposed for the first time. Several ILs with different cations and anions and the microwave-assisted extraction (MAE conditions, including sample particle size, extraction time and liquid-solid ratio, were investigated. Two M 1-butyl-3-methylimidazolium bromide ([bmim] Br solution with 0.80 M HCl was selected as the optimal solvent. Meanwhile the optimized conditions a ratio of liquid to material of 30:1, and the extraction for 10 min at 70 °C. Compared with conventional heat-reflux extraction (CHRE and the regular MAE, IL-MAE exhibited a higher extraction yield and shorter extraction time (from 1.5 h to 10 min. The optimized extraction samples were analysed by LC-MS/MS. IL extracts of Bauhinia championii (Benth.Benth consisted mainly of flavonoids, among which myricetin, quercetin and kaempferol, β-sitosterol, triacontane and hexacontane were identified. The study indicated that IL-MAE was an efficient and rapid method with simple sample preparation. LC-MS/MS was also used to determine the chemical composition of the ethyl acetate/MAE extract of Bauhinia championii (Benth. Benth, and it maybe become a rapid method to determine the composition of new plant extracts.

  16. Survey of Region-Based Text Extraction Techniques for Efficient Indexing of Image/Video Retrieval

    OpenAIRE

    Samabia Tehsin; Asif Masood; Sumaira Kausar

    2014-01-01

    With the dramatic increase in multimedia data, escalating trend of internet, and amplifying use of image/video capturing devices; content based indexing and text extraction is gaining more and more importance in research community. In the last decade, many techniques for text extraction are reported in the literature. Methodologies of text extraction from images/videos is generally comprises of text detection and localization, text tracking, text segmentation and optical character recognition...

  17. Effect of plant extracts formulated in different ointment bases on MDR strains

    OpenAIRE

    Pawar Pallavi; Nabar Bela

    2010-01-01

    Extracts of Aloe vera whole plant, Eucalyptus globulus leaves, Ficus infectoria bark, Ficus religiosa bark and Piper betel leaves were studied for antibacterial activity on resistant and sensitive strains, isolated from skin and soft tissue infections. A combination of hot alcoholic extracts of Ficus infectoria, Ficus religiosa and Piper betel were found to be more effective against all the isolates. The combined extract was formulated in different ointment bases such as polyethylene glycol, ...

  18. Technology of drinks based on water extracts of rosehip, sea buckthorn and viburnum

    OpenAIRE

    Капліна, Тетяна Вікторівна; Миронов, Денис Анатольевич

    2014-01-01

    The results of developing the technology of drinks, fruit drinks and fizzes, made of plant extracts of rosehip, sea buckthorn and viburnum, previously processed in the vortex layer of ferromagnetic particles are presented in the paper. Plant extracts were prepared by the traditional infusion at a constant temperature. Processing in the vortex layer of ferromagnetic particles allows to reduce the infusion time by 4-6 times in comparison with the industrial extraction technologies.Based on the ...

  19. Transcriptomics in the tropics: Total RNA-based profiling of Costa Rican bromeliad-associated communities

    Directory of Open Access Journals (Sweden)

    Shana K. Goffredi

    2015-01-01

    Full Text Available RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments (‘tanks’ formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24, compared to only 14 phyla in the other (Wg25. Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size (i.e. vertical oxygen gradient, and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2. Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length, genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts.

  20. Transcriptomics in the tropics: Total RNA-based profiling of Costa Rican bromeliad-associated communities.

    Science.gov (United States)

    Goffredi, Shana K; Jang, Gene E; Haroon, Mohamed F

    2015-01-01

    RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments ('tanks') formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24), compared to only 14 phyla in the other (Wg25). Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size (i.e. vertical oxygen gradient), and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2). Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length), genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts. PMID:25755850

  1. Mutation rates and evolution of multiple coding in RNA-based protocells.

    Science.gov (United States)

    de Boer, Folkert K; Hogeweg, Paulien

    2014-12-01

    RNA has a myriad of biological roles in contemporary life. We use the RNA paradigm for genotype-phenotype mappings to study the evolution of multiple coding in dependence to mutation rates. We study three different one-to-many genotype-phenotype mappings which have the potential to encode the information for multiple functions on a single sequence. These three different maps are (i) cofolding, where two sequences can bind and "cofold," (ii) suboptimal folding, where the alternative foldings within a certain range of the native state of sequences are considered, and (iii) adapter-based folding, in which protocells can evolve adapter-mediated alternative foldings. We study how protocells with a set of sequences can code for a set of predefined functional structures, while avoiding all other structures, which are considered to be misfoldings. Note that such misfolded structures are far more prevalent than functional ones. Our results highlight the flexibility of the RNA sequence to secondary structure mapping and the power of evolution to shape the genotype-phenotype mapping. We show that high fitness can be achieved even at high mutation rates. Mutation rates affect genome size, but differently depending on which folding method is used. We observe that cofolding limits the possibility to avoid misfolded structures and that adapters are always beneficial for fitness, but even more beneficial at low mutation rates. In all cases, the evolution procedure selects for molecules that can form additional structures. Our results indicate that inherent properties of RNA molecules and their interactions allow the evolution of complexity even at high mutation rates. PMID:25280530

  2. HTML Extraction Algorithm Based on Property and Data Cell

    Science.gov (United States)

    Purnamasari, Detty; Wayan Simri Wicaksana, I.; Harmanto, Suryadi; Yuniar Banowosari, Lintang

    2013-06-01

    The data available on the Internet is in various models and formats. One form of data representation is a table. Tables extraction is used in process more than one table on the Internet from different sources. Currently the effort is done by using copy-paste that is not automatic process. This article presents an approach to prepare the area, so tables in HTML format can be extracted and converted into a database that make easier to combine the data from many resources. This article was tested on the algorithm 1 used to determine the actual number of columns and rows of the table, as well as algorithm 2 are used to determine the boundary line of the property. Tests conducted at 100 tabular HTML format, and the test results provide the accuracy of the algorithm 1 is 99.9% and the accuracy of the algorithm 2 is 84%.

  3. Performance Analysis of Vision-Based Deep Web Data Extraction for Web Document Clustering

    Directory of Open Access Journals (Sweden)

    M. Lavanya

    2013-01-01

    Full Text Available Web Data Extraction is a critical task by applying various scientific tools and in a broad range of application domains. To extract data from multiple web sites are becoming more obscure, as well to design of web information extraction systems becomes more complex and time-consuming. We also present in this paper so far various risks in web data extraction. Identifying data region from web is a noteworthy crisis for information extraction from the web page. In this paper, performance of vision-based deep web data extraction for web document clustering is presented with experimental result. The proposed approach comprises of two phases: 1 Vision-based web data extraction, where output of phase I is given to second phase and 2 web document clustering. In phase 1, the web page information is segmented into various chunks. From which, surplus noise and duplicate chunks are removed using three parameters, such as hyperlink percentage, noise score and cosine similarity. To identify the relevant chunk, three parameters such as Title word Relevancy, Keyword frequency-based chunk selection, Position features are used and then, a set of keywords are extracted from those main chunks. Finally, the extracted keywords are subjected to web document clustering using Fuzzy c-means clustering (FCM. The experimentation has been performed on two different datasets and the results showed that the proposed VDEC method can achieve stable and good results of about 99.2% and 99.1% precision value in both datasets.

  4. Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Antonysamy, Stephen S.; Aubol, Brandon; Blaney, Jeff; Browner, Michelle F.; Giannetti, Anthony M.; Harris, Seth F.; Hébert, Normand; Hendle, Jörg; Hopkins, Stephanie; Jefferson, Elizabeth; Kissinger, Charles; Leveque, Vincent; Marciano, David; McGee, Ethel; Nájera, Isabel; Nolan, Brian; Tomimoto, Masaki; Torres, Eduardo; Wright, Tobi (SGX); (Roche)

    2009-07-22

    Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with {approx}1-10 mM binding affinity (K{sub D}) were iteratively optimized to give leads with {approx}200 nM biochemical activity and low {micro}M cellular activity in a Replicon assay.

  5. Essential RNA-Based Technologies and Their Applications in Plant Functional Genomics.

    Science.gov (United States)

    Teotia, Sachin; Singh, Deepali; Tang, Xiaoqing; Tang, Guiliang

    2016-02-01

    Genome sequencing has not only extended our understanding of the blueprints of many plant species but has also revealed the secrets of coding and non-coding genes. We present here a brief introduction to and personal account of key RNA-based technologies, as well as their development and applications for functional genomics of plant coding and non-coding genes, with a focus on short tandem target mimics (STTMs), artificial microRNAs (amiRNAs), and CRISPR/Cas9. In addition, their use in multiplex technologies for the functional dissection of gene networks is discussed. PMID:26774589

  6. Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips.

    Science.gov (United States)

    Wei, Zewen; Li, Zhihong

    2014-01-01

    Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h.

  7. A green deep eutectic solvent-based aqueous two-phase system for protein extracting

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Kaijia; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Huang, Yanhua; Li, Na; Wen, Qian

    2015-03-15

    Highlights: • A strategy for the protein purification with a deep eutectic solvent(DES)-based aqueous two-phase system. • Choline chloride-glycerin DES was selected as the extraction solvent. • Bovine serum albumin and trypsin were used as the analytes. • Aggregation phenomenon was detected in the mechanism research. - Abstract: As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.

  8. A green deep eutectic solvent-based aqueous two-phase system for protein extracting

    International Nuclear Information System (INIS)

    Highlights: • A strategy for the protein purification with a deep eutectic solvent(DES)-based aqueous two-phase system. • Choline chloride-glycerin DES was selected as the extraction solvent. • Bovine serum albumin and trypsin were used as the analytes. • Aggregation phenomenon was detected in the mechanism research. - Abstract: As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins

  9. Covariant Evolutionary Event Analysis for Base Interaction Prediction Using a Relational Database Management System for RNA.

    Science.gov (United States)

    Xu, Weijia; Ozer, Stuart; Gutell, Robin R

    2009-01-01

    With an increasingly large amount of sequences properly aligned, comparative sequence analysis can accurately identify not only common structures formed by standard base pairing but also new types of structural elements and constraints. However, traditional methods are too computationally expensive to perform well on large scale alignment and less effective with the sequences from diversified phylogenetic classifications. We propose a new approach that utilizes coevolutional rates among pairs of nucleotide positions using phylogenetic and evolutionary relationships of the organisms of aligned sequences. With a novel data schema to manage relevant information within a relational database, our method, implemented with a Microsoft SQL Server 2005, showed 90% sensitivity in identifying base pair interactions among 16S ribosomal RNA sequences from Bacteria, at a scale 40 times bigger and 50% better sensitivity than a previous study. The results also indicated covariation signals for a few sets of cross-strand base stacking pairs in secondary structure helices, and other subtle constraints in the RNA structure. PMID:20502534

  10. The Automatic Generation of Chinese Outline Font Based on Stroke Extraction

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    A new method to obtain spline outline description of Chinese font based on stroke extraction is presented.It has two primary advantages:(1)the quality of Chinese output is greatly improved;(2)the memory requirement is reduced.The method for stroke extraction is discussed in detail and experimental results are presented.

  11. Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling Amplification Based on Aggregation-Induced Emission Luminogens.

    Science.gov (United States)

    Min, Xuehong; Zhang, Mengshi; Huang, Fujian; Lou, Xiaoding; Xia, Fan

    2016-04-13

    Enzyme-assisted detection strategies of microRNAs (miRNAs) in vitro have accomplished both great sensitivity and specificity. However, low expression of miRNAs and a complex environment in cells induces big challenges for monitoring and tracking miRNAs in vivo. The work reports the attempt to carry miRNA imaging into live cells, by enzyme-aided recycling amplification. We utilize facile probes based yellow aggregation-induced emission luminogens (AIEgens) with super photostable property but without quencher, which are applied to monitor miRNAs not only from urine sample extracts (in vitro) but also in live cells (in vivo). The assay could distinguish the cancer patients' urine samples from the healthy urine due to the good specificity. Moreover, the probe showed much higher fluorescence intensity in breast cancer cells (MCF-7) (miR-21 in high expression) than that in cervical cancer cells (HeLa) and human lung fibroblast cells (HLF) (miR-21 in low expression) in more than 60 min, which showed the good performance and super photostability for the probe in vivo. As controls, another two probes with FAM/Cy3 and corresponding quenchers, respectively, could perform miRNAs detections in vitro and parts of in vivo tests but were not suitable for the long-term cell tracking due to the photobleach phenomena, which also demonstrates that the probe with AIEgens is a potential candidate for the accurate identification of cancer biomarkers.

  12. Optical Sensing Material for pH Detection based on the Use of Roselle Extract

    International Nuclear Information System (INIS)

    This research assessed the potential of natural colour extract of Hibiscus Sabdariffa L. (roselle) as sensing material.The pH sensor was developed based on the use of natural reddish colour in roselle calyx, delphinidin-3-sambubioside immobilised in a glass fibre filter paper. In free solution, roselle extract was characterised by using UV-visible spectrophotometer to study the effect of pH, extract concentration, response time, repeatability and photo stability. The study showed that natural colour extract can be used as sensing material for the development of an optical pH sensor. (author)

  13. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome

    Science.gov (United States)

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong

    2016-01-01

    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells.

  14. NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles.

    Science.gov (United States)

    Yang, Yanmei; Liu, Fang; Liu, Xiaogang; Xing, Bengang

    2013-01-01

    The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, "naked" siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface could be cleaved by the upconverted UV light and thus initiated the intracellular release of the siRNA. The in vitro agarose gel electrophoresis and intracellular imaging results indicated that the Si-UCNPs-based gene carrier system allowed effective siRNA delivery and the applications of NIR light instead of direct high energy UV irradiation may greatly guarantee less cell damage. PMID:23154830

  15. Corner Feature Extraction: Techniques for Landmark Based Navigation Systems

    OpenAIRE

    Namoshe, Molaletsa; Matsebe, Oudetse; Tlale, Nkgatho

    2010-01-01

    In this paper we discussed the results of an EKF SLAM using real data logged and computed offline. One of the most important parts of the SLAM process is to accurately map the environment the robot is exploring and localize in it. To achieve this however, is depended on the precise acquirement of features extracted from the external sensor. We looked at corner detection methods and we proposed an improved version of the method discussed in section 2.1.1. It transpired that methods found in th...

  16. Subpixel accuracy for extracting groove center based on corner detection

    Institute of Scientific and Technical Information of China (English)

    Liu Suyi; Wang Guorong; Shi Yonghua

    2006-01-01

    Subpixel accuracy for V-groove center in robot welding is researched and a software measure to increase the accuracy of seam tracking by laser is presented.LOG( Laplacian of Gaussian ) operator is adopted to detect image edge.Vgroove center is extracted by corner detection of extremum curvature.Subpixel position is obtained by Lagarange polynomial interpolation algorithm.Experiment results show that the method is brief and applied, and is sufficient for the real time of robot welding by laser sensors.

  17. Hardware Prototyping of Neural Network based Fetal Electrocardiogram Extraction

    Science.gov (United States)

    Hasan, M. A.; Reaz, M. B. I.

    2012-01-01

    The aim of this paper is to model the algorithm for Fetal ECG (FECG) extraction from composite abdominal ECG (AECG) using VHDL (Very High Speed Integrated Circuit Hardware Description Language) for FPGA (Field Programmable Gate Array) implementation. Artificial Neural Network that provides efficient and effective ways of separating FECG signal from composite AECG signal has been designed. The proposed method gives an accuracy of 93.7% for R-peak detection in FHR monitoring. The designed VHDL model is synthesized and fitted into Altera's Stratix II EP2S15F484C3 using the Quartus II version 8.0 Web Edition for FPGA implementation.

  18. FEATURE EXTRACTION OF BONES AND SKIN BASED ON ULTRASONIC SCANNING

    Institute of Scientific and Technical Information of China (English)

    Zheng Shuxian; Zhao Wanhua; Lu Bingheng; Zhao Zhao

    2005-01-01

    In the prosthetic socket design, aimed at the high cost and radiation deficiency caused by CT scanning which is a routine technique to obtain the cross-sectional image of the residual limb, a new ultrasonic scanning method is developed to acquire the bones and skin contours of the residual limb. Using a pig fore-leg as the scanning object, an overlapping algorithm is designed to reconstruct the 2D cross-sectional image, the contours of the bone and skin are extracted using edge detection algorithm and the 3D model of the pig fore-leg is reconstructed by using reverse engineering technology. The results of checking the accuracy of the image by scanning a cylinder work pieces show that the extracted contours of the cylinder are quite close to the standard circumference. So it is feasible to get the contours of bones and skin by ultrasonic scanning. The ultrasonic scanning system featuring no radiation and low cost is a kind of new means of cross section scanning for medical images.

  19. Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP.

    Science.gov (United States)

    Wang, Xin C; Wilson, Stephen C; Hammond, Ming C

    2016-09-30

    Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host-microbial and microbial-microbial interactions through small molecule signals. PMID:27382070

  20. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  1. A novel murmur-based heart sound feature extraction technique using envelope-morphological analysis

    Science.gov (United States)

    Yao, Hao-Dong; Ma, Jia-Li; Fu, Bin-Bin; Wang, Hai-Yang; Dong, Ming-Chui

    2015-07-01

    Auscultation of heart sound (HS) signals serves as an important primary approach to diagnose cardiovascular diseases (CVDs) for centuries. Confronting the intrinsic drawbacks of traditional HS auscultation, computer-aided automatic HS auscultation based on feature extraction technique has witnessed explosive development. Yet, most existing HS feature extraction methods adopt acoustic or time-frequency features which exhibit poor relationship with diagnostic information, thus restricting the performance of further interpretation and analysis. Tackling such a bottleneck problem, this paper innovatively proposes a novel murmur-based HS feature extraction method since murmurs contain massive pathological information and are regarded as the first indications of pathological occurrences of heart valves. Adapting discrete wavelet transform (DWT) and Shannon envelope, the envelope-morphological characteristics of murmurs are obtained and three features are extracted accordingly. Validated by discriminating normal HS and 5 various abnormal HS signals with extracted features, the proposed method provides an attractive candidate in automatic HS auscultation.

  2. a Probability-Based Statistical Method to Extract Water Body of TM Images with Missing Information

    Science.gov (United States)

    Lian, Shizhong; Chen, Jiangping; Luo, Minghai

    2016-06-01

    Water information cannot be accurately extracted using TM images because true information is lost in some images because of blocking clouds and missing data stripes, thereby water information cannot be accurately extracted. Water is continuously distributed in natural conditions; thus, this paper proposed a new method of water body extraction based on probability statistics to improve the accuracy of water information extraction of TM images with missing information. Different disturbing information of clouds and missing data stripes are simulated. Water information is extracted using global histogram matching, local histogram matching, and the probability-based statistical method in the simulated images. Experiments show that smaller Areal Error and higher Boundary Recall can be obtained using this method compared with the conventional methods.

  3. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    Energy Technology Data Exchange (ETDEWEB)

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  4. Selecting molecular therapeutic drug targets based on the expression profiles of intrahepatic cholangiocarcinomas and miRNA-mRNA regulatory networks.

    Science.gov (United States)

    Sun, Boshi; Xie, Changming; Zheng, Tongsen; Yin, Dalong; Wang, Jiabei; Liang, Yingjian; Li, Yuejin; Yang, Guangchao; Shi, Huawen; Pei, Tiemin; Han, Jihua; Liu, Lianxin

    2016-01-01

    The incidence of intrahepatic cholangiocarcinoma (ICC) is increasing yearly, making it the second most common carcinoma after hepatocellular carcinoma among primary malignant liver tumors. Integrated miRNA and mRNA analysis is becoming more frequently used in antitumor ICC treatment. However, this approach generates vast amounts of data, which leads to difficulties performing comprehensive analyses to identify specific therapeutic drug targets. In this study, we provide an in-depth analysis of ICC function, identifying potential highly potent antitumor drugs for antitumor therapy. Two sets of whole genome expression profiles were obtained from the Gene Expression Omnibus (GEO) database. Using modular bioinformatic analysis, six core functional modules were identified for ICC. Based on a Fisher's test of the Cmap small molecule drug database, 65 drug components were identified that regulated the genes of these six core modules. Literature mining was then used to identify 15 new potential antitumor drugs. PMID:26498995

  5. RCK: accurate and efficient inference of sequence- and structure-based protein–RNA binding models from RNAcompete data

    Science.gov (United States)

    Orenstein, Yaron; Wang, Yuhao; Berger, Bonnie

    2016-01-01

    Motivation: Protein–RNA interactions, which play vital roles in many processes, are mediated through both RNA sequence and structure. CLIP-based methods, which measure protein–RNA binding in vivo, suffer from experimental noise and systematic biases, whereas in vitro experiments capture a clearer signal of protein RNA-binding. Among them, RNAcompete provides binding affinities of a specific protein to more than 240 000 unstructured RNA probes in one experiment. The computational challenge is to infer RNA structure- and sequence-based binding models from these data. The state-of-the-art in sequence models, Deepbind, does not model structural preferences. RNAcontext models both sequence and structure preferences, but is outperformed by GraphProt. Unfortunately, GraphProt cannot detect structural preferences from RNAcompete data due to the unstructured nature of the data, as noted by its developers, nor can it be tractably run on the full RNACompete dataset. Results: We develop RCK, an efficient, scalable algorithm that infers both sequence and structure preferences based on a new k-mer based model. Remarkably, even though RNAcompete data is designed to be unstructured, RCK can still learn structural preferences from it. RCK significantly outperforms both RNAcontext and Deepbind in in vitro binding prediction for 244 RNAcompete experiments. Moreover, RCK is also faster and uses less memory, which enables scalability. While currently on par with existing methods in in vivo binding prediction on a small scale test, we demonstrate that RCK will increasingly benefit from experimentally measured RNA structure profiles as compared to computationally predicted ones. By running RCK on the entire RNAcompete dataset, we generate and provide as a resource a set of protein–RNA structure-based models on an unprecedented scale. Availability and Implementation: Software and models are freely available at http://rck.csail.mit.edu/ Contact: bab@mit.edu Supplementary information

  6. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque

    Directory of Open Access Journals (Sweden)

    Mahendra Jaideep

    2010-01-01

    Full Text Available Background: Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known. Aim: The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease. Materials and Methods: Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction. Statistical Analysis Used: Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis. Results: Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples. Conclusions: Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.

  7. Maximizing Expected Base Pair Accuracy in RNA Secondary Structure Prediction by Joining Stochastic Context-Free Grammars Method

    Directory of Open Access Journals (Sweden)

    Shahira M. Habashy

    2012-03-01

    Full Text Available The identification of RNA secondary structures has been among the most exciting recent developments in biology and medical science. Prediction of RNA secondary structure is a fundamental problem in computational structural biology. For several decades, free energy minimization has been the most popular method for prediction from a single sequence. It is based on a set of empirical free energy change parameters derived from experiments using a nearest-neighbor model. Accurate prediction of RNA secondary structure from the base sequence is an unsolved computational challenge. The accuracy of predictions made by free energy minimization is limited by the quality of the energy parameters in the underlying free energy model. More recently, stochastic context-free grammars (SCFGs have emerged as an alternative probabilistic methodology for modeling RNA structure. Unlike physics-based methods, which rely on thousands of experimentally -measured thermodynamic parameters, SCFGs use fully-automated statistical learning algorithms to derive model parameters. This paper proposes a new algorithm that computes base pairing pattern for RNA molecule. Complex internal structures in RNA are fully taken into account. It supports the calculation of stochastic context-free grammars (SCFGs, and equilibrium concentrations of duplex structures. This new algorithm is compared with dynamic programming benchmark mfold and algorithms (Tfold, and MaxExpect. The results showed that the proposed algorithm achieved better performance with respect to sensitivity and positive predictive value.

  8. An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes.

    Science.gov (United States)

    Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Masaki, Shunpei; Nakayama, Hiroshi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki

    2009-11-01

    We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a approximately 21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

  9. Texture based feature extraction methods for content based medical image retrieval systems.

    Science.gov (United States)

    Ergen, Burhan; Baykara, Muhammet

    2014-01-01

    The developments of content based image retrieval (CBIR) systems used for image archiving are continued and one of the important research topics. Although some studies have been presented general image achieving, proposed CBIR systems for archiving of medical images are not very efficient. In presented study, it is examined the retrieval efficiency rate of spatial methods used for feature extraction for medical image retrieval systems. The investigated algorithms in this study depend on gray level co-occurrence matrix (GLCM), gray level run length matrix (GLRLM), and Gabor wavelet accepted as spatial methods. In the experiments, the database is built including hundreds of medical images such as brain, lung, sinus, and bone. The results obtained in this study shows that queries based on statistics obtained from GLCM are satisfied. However, it is observed that Gabor Wavelet has been the most effective and accurate method. PMID:25227014

  10. Validation of artificial microRNA expression by poly(A) tailing-based RT-PCR

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Rui Shi, Chenmin Yang, Ronald Sederoff & Vincent Chiang ### Abstract Here we describe a protocol for validating expression of artificial microRNAs (amiRNAs) by poly(A) tailing-based RT-PCR. Total RNAs, including amiRNA, are poly(A) tailed using E.coli. poly(A) polymerase. Poly(A) tailed amiRNA can be converted into cDNA along with mRNAs in a reverse transcription reaction primed by a standard poly(T) anchor adaptor. AmiRNA can then be amplified and quantitated by real-tim...

  11. Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

    DEFF Research Database (Denmark)

    Askou, Anne Louise; Aagaard, Lars; Kostic, Corinne;

    2015-01-01

    Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters...... by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new...

  12. Clinical performance of the new Roche COBAS (R) TaqMan HCV test and high pure system for extraction, detection and quantitation of HCV RNA in plasma and serum

    NARCIS (Netherlands)

    H.C. Gelderblom; S. Menting; M.G. Beld

    2006-01-01

    We evaluated the Roche COBAS (R) TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported li

  13. A Method of SAR Target Recognition Based on Gabor Filter and Local Texture Feature Extraction

    OpenAIRE

    Wang Lu; Zhang Fan; Li Wei; Xie Xiao-ming; Hu Wei

    2015-01-01

    This paper presents a novel texture feature extraction method based on a Gabor filter and Three-Patch Local Binary Patterns (TPLBP) for Synthetic Aperture Rader (SAR) target recognition. First, SAR images are processed by a Gabor filter in different directions to enhance the significant features of the targets and their shadows. Then, the effective local texture features based on the Gabor filtered images are extracted by TPLBP. This not only overcomes the shortcoming of Local Binary Patterns...

  14. Post-processing of Deep Web Information Extraction Based on Domain Ontology

    OpenAIRE

    Peng, T.; L. Liu

    2013-01-01

    Many methods are utilized to extract and process query results in deep Web, which rely on the different structures of Web pages and various designing modes of databases. However, some semantic meanings and relations are ignored. So, in this paper, we present an approach for post-processing deep Web query results based on domain ontology which can utilize the semantic meanings and relations. A block identification model (BIM) based on node similarity is defined to extract data blocks that ...

  15. SATEN: An Object-Oriented Web-Based Revision and Extraction Engine

    OpenAIRE

    Williams, Mary-Anne; Sims, Aidan

    2000-01-01

    SATEN is an object-oriented web-based extraction and belief revision engine. It runs on any computer via a Java 1.1 enabled browser such as Netscape 4. SATEN performs belief revision based on the AGM approach. The extraction and belief revision reasoning engines operate on a user specified ranking of information. One of the features of SATEN is that it can be used to integrate mutually inconsistent commensuate rankings into a consistent ranking.

  16. Iris Feature Extraction Method Based on 1D Gabor Filter

    Institute of Scientific and Technical Information of China (English)

    XU Guang-zhu; MA Yi-de; ZHANG Zai-feng

    2008-01-01

    The normalized iris image was divided into eight sub-bands, and every column of each sub-band was averaged by rows to generate eight 1D iris signals. Then the even symmetry item of 1D Gabor filter was used to describe local characteristic blocks in 1D iris signals, and the results were quantified by their polarities to generate iris codes. In order to estimate the performance of the presented method, an iris recognition platform was produced and the Hamming distance between two iris codes was computed to measure the dissimilarity of them. The experimental results in CASIA v1 0 and Bath iris image databases show that the proposed iris feature extraction algorithm has a promising potential in iris recognition.

  17. Image-based mobile service: automatic text extraction and translation

    Science.gov (United States)

    Berclaz, Jérôme; Bhatti, Nina; Simske, Steven J.; Schettino, John C.

    2010-01-01

    We present a new mobile service for the translation of text from images taken by consumer-grade cell-phone cameras. Such capability represents a new paradigm for users where a simple image provides the basis for a service. The ubiquity and ease of use of cell-phone cameras enables acquisition and transmission of images anywhere and at any time a user wishes, delivering rapid and accurate translation over the phone's MMS and SMS facilities. Target text is extracted completely automatically, requiring no bounding box delineation or related user intervention. The service uses localization, binarization, text deskewing, and optical character recognition (OCR) in its analysis. Once the text is translated, an SMS message is sent to the user with the result. Further novelties include that no software installation is required on the handset, any service provider or camera phone can be used, and the entire service is implemented on the server side.

  18. Feature evaluation and extraction based on neural network in analog circuit fault diagnosis

    Institute of Scientific and Technical Information of China (English)

    Yuan Haiying; Chen Guangju; Xie Yongle

    2007-01-01

    Choosing the right characteristic parameter is the key to fault diagnosis in analog circuit.The feature evaluation and extraction methods based on neural network are presented.Parameter evaluation of circuit features is realized by training results from neural network; the superior nonlinear mapping capability is competent for extracting fault features which are normalized and compressed subsequently.The complex classification problem on fault pattern recognition in analog circuit is transferred into feature processing stage by feature extraction based on neural network effectively, which improves the diagnosis efficiency.A fault diagnosis illustration validated this method.

  19. Feature Fusion Based Road Extraction for HJ-1-C SAR Image

    OpenAIRE

    Lu Ping-ping; Du Kang-ning; Yu Wei-dong; Wang Yu; Deng Yun-kai

    2014-01-01

    Road network extraction in SAR images is one of the key tasks of military and civilian technologies. To solve the issues of road extraction of HJ-1-C SAR images, a road extraction algorithm is proposed based on the integration of ratio and directional information. Due to the characteristic narrow dynamic range and low signal to noise ratio of HJ-1-C SAR images, a nonlinear quantization and an image filtering method based on a multi-scale autoregressive model are proposed here. A road extracti...

  20. Monoolein-based nanocarriers for enhanced folate receptor-mediated RNA delivery to cancer cells.

    Science.gov (United States)

    Lopes, Ivo; C N Oliveira, Ana; P Sárria, Marisa; P Neves Silva, João; Gonçalves, Odete; Gomes, Andreia C; Real Oliveira, Maria Elisabete C D

    2016-09-01

    We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and the helper lipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25 µg/mL of lipid and 31% for 40 µg/mL) when compared with folate receptor α negative cells (31% for 25 µg/mL of lipid and 23% for 40 µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest. PMID:26340109

  1. Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

    Science.gov (United States)

    Lee, Jonghwan; Choi, Kyung-Ju; Moon, Sung Ung; Kim, Soonhag

    2016-01-01

    Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs.

  2. Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

    Science.gov (United States)

    Lee, Jonghwan; Choi, Kyung-Ju; Moon, Sung Ung; Kim, Soonhag

    2016-01-01

    Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs. PMID:26454049

  3. A Method of Road Extraction from High-resolution Remote Sensing Images Based on Shape Features

    Directory of Open Access Journals (Sweden)

    LEI Xiaoqi

    2016-02-01

    Full Text Available Road extraction from high-resolution remote sensing image is an important and difficult task.Since remote sensing images include complicated information,the methods that extract roads by spectral,texture and linear features have certain limitations.Also,many methods need human-intervention to get the road seeds(semi-automatic extraction,which have the great human-dependence and low efficiency.The road-extraction method,which uses the image segmentation based on principle of local gray consistency and integration shape features,is proposed in this paper.Firstly,the image is segmented,and then the linear and curve roads are obtained by using several object shape features,so the method that just only extract linear roads are rectified.Secondly,the step of road extraction is carried out based on the region growth,the road seeds are automatic selected and the road network is extracted.Finally,the extracted roads are regulated by combining the edge information.In experiments,the images that including the better gray uniform of road and the worse illuminated of road surface were chosen,and the results prove that the method of this study is promising.

  4. MicroRNA-19a/b mediates grape seed procyanidin extract-induced anti-neoplastic effects against lung cancer.

    Science.gov (United States)

    Mao, Jenny T; Xue, Bingye; Smoake, Jane; Lu, Qing-Yi; Park, Heesung; Henning, Susanne M; Burns, Windie; Bernabei, Alvise; Elashoff, David; Serio, Kenneth J; Massie, Larry

    2016-08-01

    Oncomirs are microRNAs (miRNA) associated with carcinogenesis and malignant transformation. They have emerged as potential molecular targets for anti-cancer therapy. We hypothesize that grape seed procyanidin extract (GSE) exerts antineoplastic effects through modulations of oncomirs and their downstream targets. We found that GSE significantly down-regulated oncomirs miR-19a and -19b in a variety of lung neoplastic cells. GSE also increased mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homolog (PTEN), both predicted targets of miR-19a and -19b. Furthermore, GSE significantly increased PTEN activity and decreased AKT phosphorylation in A549 cells. Transfection of miR-19a and -19b mimics reversed the up-regulations of IGF2R and PTEN gene expression and abrogated the GSE induced anti-proliferative response. Additionally, oral administration of leucoselect phytosome, comprised of standardized grape seed oligomeric procyanidins complexed with soy phospholipids, to athymic nude mice via gavage, significantly down-regulated miR-19a, -19b and the miR-17-92 cluster host gene (MIR17HG) expressions, increased IGF-2R, PTEN, decreased phosphorylated-AKT in A549 xenograft tumors, and markedly inhibited tumor growth. To confirm the absorption of orally administered GSE, plasma procyanidin B1 levels, between 60 and 90 min after gavage of leucoselect phytosome (400 mg/kg), were measured by LC/MS at week 2 and 8 of treatment; the estimated concentration that was associated with 50% growth inhibition (IC50) (1.3 μg/mL) in vitro was much higher than the IC50 (0.032-0.13 μg/ml) observed in vivo. Our findings reveal novel antineoplastic mechanisms by GSE and support the clinical translation of leucoselect phytosome as an anti-neoplastic and chemopreventive agent for lung cancer. PMID:27289489

  5. Analysis of the contributions of ring current and electric field effects to the chemical shifts of RNA bases.

    Science.gov (United States)

    Sahakyan, Aleksandr B; Vendruscolo, Michele

    2013-02-21

    Ring current and electric field effects can considerably influence NMR chemical shifts in biomolecules. Understanding such effects is particularly important for the development of accurate mappings between chemical shifts and the structures of nucleic acids. In this work, we first analyzed the Pople and the Haigh-Mallion models in terms of their ability to describe nitrogen base conjugated ring effects. We then created a database (DiBaseRNA) of three-dimensional arrangements of RNA base pairs from X-ray structures, calculated the corresponding chemical shifts via a hybrid density functional theory approach and used the results to parametrize the ring current and electric field effects in RNA bases. Next, we studied the coupling of the electric field and ring current effects for different inter-ring arrangements found in RNA bases using linear model fitting, with joint electric field and ring current, as well as only electric field and only ring current approximations. Taken together, our results provide a characterization of the interdependence of ring current and electric field geometric factors, which is shown to be especially important for the chemical shifts of non-hydrogen atoms in RNA bases.

  6. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    Science.gov (United States)

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

  7. SAW: a method to identify splicing events from RNA-Seq data based on splicing fingerprints.

    Directory of Open Access Journals (Sweden)

    Kang Ning

    Full Text Available Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons.

  8. DNA/RNA transverse current sequencing: Intrinsic structural noise from neighboring bases

    Directory of Open Access Journals (Sweden)

    Jose eAlvarez

    2015-06-01

    Full Text Available Nanopore DNA sequencing via transverse current has emerged as a promising candidate for third-generation sequencing technology. It produces long read lengths which could alleviate problems with assembly errors inherent in current technologies. However, the high error rates of nanopore sequencing have to be addressed. A very important source of the error is the intrinsic noise in the current arising from carrier dispersion along the chain of the molecule i.e. from the influence of neighboring bases. In this work we perform calculations of the transverse current within an effective multi-orbital tight-binding model derived from first-principles calculations of the DNA/RNA molecules, to study the effect of this structural noise on the error rates in DNA/RNA sequencing via transverse current in nanopores. We demonstrate that a statistical technique, utilizing not only the currents through the nucleotides but also the correlations in the currents, can in principle reduce the error rate below any desired precision.

  9. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts

    Science.gov (United States)

    Huh, Christine J; Zhang, Bo; Victor, Matheus B; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; Yoo, Andrew S

    2016-01-01

    Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion. DOI: http://dx.doi.org/10.7554/eLife.18648.001 PMID:27644593

  10. Improved External Base Resistance Extraction for Submicrometer InP/InGaAs DHBT Models

    DEFF Research Database (Denmark)

    Johansen, Tom Keinicke; Krozer, Viktor; Nodjiadjim, Virginie;

    2011-01-01

    An improved direct parameter extraction method is proposed for III–V heterojunction bipolar transistor (HBT) external base resistance $R_{\\rm bx}$ extraction from forward active $S$-parameters. The method is formulated taking into account the current dependence of the intrinsic base–collector cap......An improved direct parameter extraction method is proposed for III–V heterojunction bipolar transistor (HBT) external base resistance $R_{\\rm bx}$ extraction from forward active $S$-parameters. The method is formulated taking into account the current dependence of the intrinsic base...... factor given as the ratio of the emitter to the collector area. The determination of the parameters $I_{p}$ and $X_{0}$ from experimental $S$-parameters is described. The method is applied to high-speed submicrometer InP/InGaAs DHBT devices and leads to small-signal equivalent circuit models, which...

  11. The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissue.

    Science.gov (United States)

    Chung, Joon-Yong; Cho, Hanbyoul; Hewitt, Stephen M

    2016-01-01

    RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investigative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue.

  12. Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.

    Science.gov (United States)

    MacFarlane, Ryan C; Singh, Upinder

    2008-06-01

    The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of "RNAi" negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem. PMID:18346737

  13. In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

    Energy Technology Data Exchange (ETDEWEB)

    Allegra, Danilo [Department of Internal Medicine III, University Hospital Ulm, Albert-Einstein-Allee 23, 89081 Ulm (Germany); Cooperation Unit ' Mechanisms of Leukemogenesis' , B061, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Mertens, Daniel, E-mail: daniel.mertens@uniklinik-ulm.de [Department of Internal Medicine III, University Hospital Ulm, Albert-Einstein-Allee 23, 89081 Ulm (Germany); Cooperation Unit ' Mechanisms of Leukemogenesis' , B061, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

    2011-03-25

    Research highlights: {yields} Posttranscriptional regulation of miRNA processing is difficult to quantify. {yields} Our in-vivo processing assay can quantify Drosha cleavage in live cells. {yields} It is based on luciferase reporters fused with pri-miRNAs. {yields} The assay validates the processing defect caused by a mutation in pri-16-1. {yields} It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.

  14. Post-processing of Deep Web Information Extraction Based on Domain Ontology

    Directory of Open Access Journals (Sweden)

    PENG, T.

    2013-11-01

    Full Text Available Many methods are utilized to extract and process query results in deep Web, which rely on the different structures of Web pages and various designing modes of databases. However, some semantic meanings and relations are ignored. So, in this paper, we present an approach for post-processing deep Web query results based on domain ontology which can utilize the semantic meanings and relations. A block identification model (BIM based on node similarity is defined to extract data blocks that are relevant to specific domain after reducing noisy nodes. Feature vector of domain books is obtained by result set extraction model (RSEM based on vector space model (VSM. RSEM, in combination with BIM, builds the domain ontology on books which can not only remove the limit of Web page structures when extracting data information, but also make use of semantic meanings of domain ontology. After extracting basic information of Web pages, a ranking algorithm is adopted to offer an ordered list of data records to users. Experimental results show that BIM and RSEM extract data blocks and build domain ontology accurately. In addition, relevant data records and basic information are extracted and ranked. The performances precision and recall show that our proposed method is feasible and efficient.

  15. Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

    Science.gov (United States)

    Sardo, C; Farra, R; Licciardi, M; Dapas, B; Scialabba, C; Giammona, G; Grassi, M; Grassi, G; Cavallaro, G

    2015-07-30

    Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models.

  16. [Classification technique for hyperspectral image based on subspace of bands feature extraction and LS-SVM].

    Science.gov (United States)

    Gao, Heng-zhen; Wan, Jian-wei; Zhu, Zhen-zhen; Wang, Li-bao; Nian, Yong-jian

    2011-05-01

    The present paper proposes a novel hyperspectral image classification algorithm based on LS-SVM (least squares support vector machine). The LS-SVM uses the features extracted from subspace of bands (SOB). The maximum noise fraction (MNF) method is adopted as the feature extraction method. The spectral correlations of the hyperspectral image are used in order to divide the feature space into several SOBs. Then the MNF is used to extract characteristic features of the SOBs. The extracted features are combined into the feature vector for classification. So the strong bands correlation is avoided and the spectral redundancies are reduced. The LS-SVM classifier is adopted, which replaces inequality constraints in SVM by equality constraints. So the computation consumption is reduced and the learning performance is improved. The proposed method optimizes spectral information by feature extraction and reduces the spectral noise. The classifier performance is improved. Experimental results show the superiorities of the proposed algorithm.

  17. Silica-Based Solid Phase Extraction of DNA on a Microchip

    Institute of Scientific and Technical Information of China (English)

    陈晓芳; 沈科跃; 刘鹏; 郭旻; 程京; 周玉祥

    2004-01-01

    Micro total analysis systems for chemical and biological analysis have attracted much attention.However,microchips for sample preparation and especially DNA purification are still underdeveloped.This work describes a solid phase extraction chip for purifying DNA from biological samples based on the adsorption of DNA on bare silica beads prepacked in a microchannel.The chip was fabricated with poly-dimethylsiloxane.The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed.Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase.The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use.The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume.Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction.

  18. Extraction of spatio-temporal information of earthquake event based on semantic technology

    Science.gov (United States)

    Fan, Hong; Guo, Dan; Li, Huaiyuan

    2015-12-01

    In this paper a web information extraction method is presented which identifies a variety of thematic events utilizing the event knowledge framework derived from text training, and then further uses the syntactic analysis to extract the event key information. The method which combines the text semantic information and domain knowledge of the event makes the extraction of information people interested more accurate. In this paper, web based earthquake news extraction is taken as an example. The paper firstly briefs the overall approaches, and then details the key algorithm and experiments of seismic events extraction. Finally, this paper conducts accuracy analysis and evaluation experiments which demonstrate that the proposed method is a promising way of hot events mining.

  19. Geometric Feature Extraction and Model Reconstruction Based on Scattered Data

    Institute of Scientific and Technical Information of China (English)

    胡鑫; 习俊通; 金烨

    2004-01-01

    A method of 3D model reconstruction based on scattered point data in reverse engineering is presented here. The topological relationship of scattered points was established firstly, then the data set was triangulated to reconstruct the mesh surface model. The curvatures of cloud data were calculated based on the mesh surface, and the point data were segmented by edge-based method; Every patch of data was fitted by quadric surface of freeform surface, and the type of quadric surface was decided by parameters automatically, at last the whole CAD model was created. An example of mouse model was employed to confirm the effect of the algorithm.

  20. Extraction and functionalization of bagasse cellulose nanofibres to Schiff-base based antimicrobial membranes.

    Science.gov (United States)

    Bansal, Monica; Chauhan, Ghanshyam S; Kaushik, Anupama; Sharma, Avantika

    2016-10-01

    The work reported in this paper involves synthesis of a nanocellulose/chitosan composite and its further modification to antimicrobial films. Bagasse, an easily available biowaste, was used as source to extract nanocellulose fibres (CNFs) by subjecting it to mechanical and chemical treatments including alkaline steam explosion and high shear homogenization. The CNFs were subjected to periodate oxidation to obtain nanocellulose dialdehyde (CDA). The aldehyde groups of CDA were reacted with amino groups of chitosan to form Schiff-base. The resulting CDA/chitosan composite fibres were characterized at various steps. The fibres were then cast into films using cellulose acetate as a binder. The films have good physical strength. The composite films show excellent antimicrobial properties when tested against Staphylococcus aureus and Escherichia coli. Such antimicrobial films have potential applications in the formation of antimicrobial packaging material. PMID:27316771

  1. Extracting RNA from Leaves of Mustard Brassica Juncea and Measurement and Analysis of Myrosinase Gene%榨菜叶中 RNA 提取及硫苷酶基因序列测定与分析

    Institute of Scientific and Technical Information of China (English)

    张燕; 侯坤兰

    2015-01-01

    采用 Trizol 试剂法从榨菜叶中提取 RNA,采用 RT-PCR 技术扩增得到硫苷酶基因保守序列,利用 DNAman7生物信息分析软件对测得的保守序列与菜心硫苷酶基因保守序列进行比对,翻译核苷酸和蛋白质序列与芸薹属油菜、菜心、甘蓝、芥菜等序列进行相应的比对。结果表明,采用 Trizol 试剂法能获得320 bp 的基因保守序列;与甘蓝、菜心硫苷酶基因保守序列比对结果的 Identity 为97.12%;与芸薹属油菜、菜心、甘蓝等进行相应的翻译核苷酸和翻译成蛋白质序列比对分析,发现序列的覆盖度与相似度均达到较高的水平。%This purposes of this research is to lay the foundation for prokaryotic expression and to improve the content and activity of myrosinase in mustard brassica juncea at the same time. The research material was taken from myrosinase gene in mustard brassica juncea. This topic get conserved sequence of myrosinase gene taken from leaves for the method used, for extraction of RNA from mustard brassica juncea leaves that Trizol reagent step separation method of total RNA, according to the conserved nucleotide sequences of different plants and conservative sequence obtained by RT -PCR, conserved sequence alignment between the obtained and Brassica rape on the DNAman7 bioinformatics analysis software, myrosinase translated nucleotides sequence and protein sequence alignment analysis online combined the NCBI and main with Brassica napue, Brassica rape, Brassica oleracea, Brassica Juncea, etc. The results shows that under identical conditions: 320bp gene conserved sequence was obtained by method of Trizol reagent step separation, bioinformatics analysis suggested that conserved sequence showed 97.12% identity to M Y B. oleracea-M, M Y 49 B. parachinensis-M, myrosinase translated nucleotides sequence and protein sequence of Mustard brassica juncea has a higher query cover and identity with Brassica napue, Brassica

  2. Extracting RNA from Leaves of Mustard Brassica Juncea and Measurement and Analysis of Myrosinase Gene%榨菜叶中 RNA 提取及硫苷酶基因序列测定与分析

    Institute of Scientific and Technical Information of China (English)

    张燕; 侯坤兰

    2015-01-01

    This purposes of this research is to lay the foundation for prokaryotic expression and to improve the content and activity of myrosinase in mustard brassica juncea at the same time. The research material was taken from myrosinase gene in mustard brassica juncea. This topic get conserved sequence of myrosinase gene taken from leaves for the method used, for extraction of RNA from mustard brassica juncea leaves that Trizol reagent step separation method of total RNA, according to the conserved nucleotide sequences of different plants and conservative sequence obtained by RT -PCR, conserved sequence alignment between the obtained and Brassica rape on the DNAman7 bioinformatics analysis software, myrosinase translated nucleotides sequence and protein sequence alignment analysis online combined the NCBI and main with Brassica napue, Brassica rape, Brassica oleracea, Brassica Juncea, etc. The results shows that under identical conditions: 320bp gene conserved sequence was obtained by method of Trizol reagent step separation, bioinformatics analysis suggested that conserved sequence showed 97.12% identity to M Y B. oleracea-M, M Y 49 B. parachinensis-M, myrosinase translated nucleotides sequence and protein sequence of Mustard brassica juncea has a higher query cover and identity with Brassica napue, Brassica rape, Brassica oleracea, etc. It found that the similarity of sequence coverage have reached a higher level.%采用 Trizol 试剂法从榨菜叶中提取 RNA,采用 RT-PCR 技术扩增得到硫苷酶基因保守序列,利用 DNAman7生物信息分析软件对测得的保守序列与菜心硫苷酶基因保守序列进行比对,翻译核苷酸和蛋白质序列与芸薹属油菜、菜心、甘蓝、芥菜等序列进行相应的比对。结果表明,采用 Trizol 试剂法能获得320 bp 的基因保守序列;与甘蓝、菜心硫苷酶基因保守序列比对结果的 Identity 为97.12%;与芸薹属油菜、菜心、甘蓝等进行相应的翻译核苷

  3. Ionic liquid-based microwave-assisted extraction of rutin from Chinese medicinal plants.

    Science.gov (United States)

    Zeng, Huan; Wang, Yuzhi; Kong, Jinhuan; Nie, Chan; Yuan, Ya

    2010-12-15

    An ionic liquid-based microwave-assisted extraction (ILMAE) method has been developed for the effective extraction of rutin from Chinese medicinal plants including Saururus chinensis (Lour.) Bail. (S. chinensis) and Flos Sophorae. A series of 1-butyl-3-methylimidazolium ionic liquids with different anions were investigated. The results indicated that the characteristics of anions have remarkable effects on the extraction efficiency of rutin and among the investigated ionic liquids, 1-butyl-3-methylimidazolium bromide ([bmim]Br) aqueous solution was the best. In addition, the ILMAE procedures for the two kinds of medicinal herbs were also optimized by means of a series of single factor experiments and an L(9) (3(4)) orthogonal design. Compared with the optimal ionic liquid-based heating extraction (ILHE), marinated extraction (ILME), ultrasonic-assisted extraction (ILUAE), the optimized approach of ILMAE gained higher extraction efficiency which is 4.879 mg/g in S. chinensis with RSD 1.33% and 171.82 mg/g in Flos Sophorae with RSD 1.47% within the shortest extraction time. Reversed phase high performance liquid chromatography (RP-HPLC) with ultraviolet detection was employed for the analysis of rutin in Chinese medicinal plants. Under the optimum conditions, the average recoveries of rutin from S. chinensis and Flos Sophorae were 101.23% and 99.62% with RSD lower than 3%, respectively. The developed approach is linear at concentrations from 42 to 252 mg L(-1) of rutin solution, with the regression coefficient (r) at 0.99917. Moreover, the extraction mechanism of ILMAE and the microstructures and chemical structures of the two researched samples before and after extraction were also investigated. With the help of LC-MS, it was future demonstrated that the two researched herbs do contain active ingredient of rutin and ionic liquids would not influence the structure of rutin.

  4. A Robust Gradient Based Method for Building Extraction from LiDAR and Photogrammetric Imagery.

    Science.gov (United States)

    Siddiqui, Fasahat Ullah; Teng, Shyh Wei; Awrangjeb, Mohammad; Lu, Guojun

    2016-01-01

    Existing automatic building extraction methods are not effective in extracting buildings which are small in size and have transparent roofs. The application of large area threshold prohibits detection of small buildings and the use of ground points in generating the building mask prevents detection of transparent buildings. In addition, the existing methods use numerous parameters to extract buildings in complex environments, e.g., hilly area and high vegetation. However, the empirical tuning of large number of parameters reduces the robustness of building extraction methods. This paper proposes a novel Gradient-based Building Extraction (GBE) method to address these limitations. The proposed method transforms the Light Detection And Ranging (LiDAR) height information into intensity image without interpolation of point heights and then analyses the gradient information in the image. Generally, building roof planes have a constant height change along the slope of a roof plane whereas trees have a random height change. With such an analysis, buildings of a greater range of sizes with a transparent or opaque roof can be extracted. In addition, a local colour matching approach is introduced as a post-processing stage to eliminate trees. This stage of our proposed method does not require any manual setting and all parameters are set automatically from the data. The other post processing stages including variance, point density and shadow elimination are also applied to verify the extracted buildings, where comparatively fewer empirically set parameters are used. The performance of the proposed GBE method is evaluated on two benchmark data sets by using the object and pixel based metrics (completeness, correctness and quality). Our experimental results show the effectiveness of the proposed method in eliminating trees, extracting buildings of all sizes, and extracting buildings with and without transparent roof. When compared with current state-of-the-art building

  5. A Robust Gradient Based Method for Building Extraction from LiDAR and Photogrammetric Imagery

    Science.gov (United States)

    Siddiqui, Fasahat Ullah; Teng, Shyh Wei; Awrangjeb, Mohammad; Lu, Guojun

    2016-01-01

    Existing automatic building extraction methods are not effective in extracting buildings which are small in size and have transparent roofs. The application of large area threshold prohibits detection of small buildings and the use of ground points in generating the building mask prevents detection of transparent buildings. In addition, the existing methods use numerous parameters to extract buildings in complex environments, e.g., hilly area and high vegetation. However, the empirical tuning of large number of parameters reduces the robustness of building extraction methods. This paper proposes a novel Gradient-based Building Extraction (GBE) method to address these limitations. The proposed method transforms the Light Detection And Ranging (LiDAR) height information into intensity image without interpolation of point heights and then analyses the gradient information in the image. Generally, building roof planes have a constant height change along the slope of a roof plane whereas trees have a random height change. With such an analysis, buildings of a greater range of sizes with a transparent or opaque roof can be extracted. In addition, a local colour matching approach is introduced as a post-processing stage to eliminate trees. This stage of our proposed method does not require any manual setting and all parameters are set automatically from the data. The other post processing stages including variance, point density and shadow elimination are also applied to verify the extracted buildings, where comparatively fewer empirically set parameters are used. The performance of the proposed GBE method is evaluated on two benchmark data sets by using the object and pixel based metrics (completeness, correctness and quality). Our experimental results show the effectiveness of the proposed method in eliminating trees, extracting buildings of all sizes, and extracting buildings with and without transparent roof. When compared with current state-of-the-art building

  6. A Robust Gradient Based Method for Building Extraction from LiDAR and Photogrammetric Imagery

    Directory of Open Access Journals (Sweden)

    Fasahat Ullah Siddiqui

    2016-07-01

    Full Text Available Existing automatic building extraction methods are not effective in extracting buildings which are small in size and have transparent roofs. The application of large area threshold prohibits detection of small buildings and the use of ground points in generating the building mask prevents detection of transparent buildings. In addition, the existing methods use numerous parameters to extract buildings in complex environments, e.g., hilly area and high vegetation. However, the empirical tuning of large number of parameters reduces the robustness of building extraction methods. This paper proposes a novel Gradient-based Building Extraction (GBE method to address these limitations. The proposed method transforms the Light Detection And Ranging (LiDAR height information into intensity image without interpolation of point heights and then analyses the gradient information in the image. Generally, building roof planes have a constant height change along the slope of a roof plane whereas trees have a random height change. With such an analysis, buildings of a greater range of sizes with a transparent or opaque roof can be extracted. In addition, a local colour matching approach is introduced as a post-processing stage to eliminate trees. This stage of our proposed method does not require any manual setting and all parameters are set automatically from the data. The other post processing stages including variance, point density and shadow elimination are also applied to verify the extracted buildings, where comparatively fewer empirically set parameters are used. The performance of the proposed GBE method is evaluated on two benchmark data sets by using the object and pixel based metrics (completeness, correctness and quality. Our experimental results show the effectiveness of the proposed method in eliminating trees, extracting buildings of all sizes, and extracting buildings with and without transparent roof. When compared with current state

  7. Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina Comparison of three different protocols for extracting RNA from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Gisele Rodrigues Gouveia

    2011-12-01

    Full Text Available INTRODUÇÃO: Tecidos fixados em formalina e emblocados em parafina (FFEP são importantes fontes de amostras para estudos retrospectivos. Apesar de sua capacidade de preservação de proteínas e morfologia celular, a formalina interfere negativamente em testes de biologia molecular por fragmentar e modificar quimicamente os ácidos nucleicos, particularmente o ácido ribonucleico (RNA. OBJETIVO: Comparar a eficiência de três diferentes protocolos de extração de RNA para análise de expressão gênica de tecidos FFEP. MATERIAL E MÉTODOS: Amostras de linfonodo humano FEEP foram submetidas à extração de RNA utilizando-se os kits Ambion e Arcturus Bioscience e o método clássico de Trizol. Após a extração, o RNA foi quantificado e testado quanto à sua capacidade de amplificaç��o pela reação em cadeia da polimerase em tempo real (RT-PCR utilizando primers do gene endógeno gliceraldeído-3 fosfato desidrogenase (GAPDH. DISCUSSÃO/CONCLUSÃO: Todos os protocolos testados produziram quantidades adequadas e suficientes de RNA total, entretanto, somente os protocolos com uso dos kits Ambion e Arcturus produziram RNA capaz de ser amplificado pela PCR.INTRODUCTION: Formalin fixed paraffin embedded (FFPE tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders Molecular Biology tests once it fragments and chemically modifies nucleic acids, particularly RNA. OBJECTIVE: To compare the efficiency of three different RNA extraction protocols for gene expression analysis of FFEP tissues. MATERIAL AND METHODS: RNA was extracted from FFPE samples of human lymph by means of Ambion and Arcturus Bioscience kits and the classical Trizol method. After extraction, RNA was quantified and tested for amplification through real time polymerase chain reaction (RT-PCR using glyceraldehydes-3 phosphate dehydrogenase (GAPDH endogenous gene primers. DISCUSSION

  8. Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

    DEFF Research Database (Denmark)

    Zhang, Guojie; Guo, Guangwu; Hu, Xueda;

    2010-01-01

    Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we...... present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative...... fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell...

  9. Participation of microRNA 124-CREB pathway: a parallel memory enhancing mechanism of standardised extract of Bacopa monniera (BESEB CDRI-08).

    Science.gov (United States)

    Preethi, Jayakumar; Singh, Hemant K; Charles, Prisila Dulcy; Rajan, Koilmani Emmanuvel

    2012-10-01

    Bacosides, the effective component of standardised leaf extract of Bacopa monniera (BESEB CDRI-08) has been reported to have memory enhancing effect. Our previous reports suggested that BESEB CDRI-08 (BME) improves memory in postnatal rats by enhancing serotonin [5-hydroxytryptamine (5-HT)] metabolism, its transportation and subsequently activates 5-HT(3A) receptor during hippocampus-dependent learning. In this study, we examine whether the up-regulated 5-HT(3A) receptor activity by BME modulate microRNA 124-CREB pathway to enhance synaptic plasticity. Wistar rat pups received single dose of vehicle solution (0.5 % gum acacia + 0.9 % saline)/BME (80 mg/kg)/mCPBG (10 mg/kg)/BME + mCPBG during the postnatal days (PND) 15-29. On PND 30, individuals were trained at brightness discrimination task and 24 h later, they were tested on the task. The BME treated group exhibited significantly lower percentage of errors during retention than acquisition. In addition, pre-miR-124 expression in hippocampus was significantly down-regulated in the BME and mCPBG + BME treated groups combined with a significant increase in the plasticity related genes, cAMP response element-binding protein, its phosphorylation and postsynaptic density protein 95. Our results suggest that this may be one of the mechanisms of bacosides present in BME for the memory enhancement. PMID:22837048

  10. Viral RNA testing and automation on the bead-based CBNE detection microsystem.

    Energy Technology Data Exchange (ETDEWEB)

    Galambos, Paul C.; Bourdon, Christopher Jay; Farrell, Cara M.; Rossito, Paul (University of California at Davis); McClain, Jaime L.; Derzon, Mark Steven; Cullor, James Sterling (University of California at Davis); Rahimian, Kamayar

    2008-09-01

    We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and

  11. MicroRNA prediction using a fixed-order Markov model based on the secondary structure pattern.

    Directory of Open Access Journals (Sweden)

    Wei Shen

    Full Text Available Predicting miRNAs is an arduous task, due to the diversity of the precursors and complexity of enzyme processes. Although several prediction approaches have reached impressive performances, few of them could achieve a full-function recognition of mature miRNA directly from the candidate hairpins across species. Therefore, researchers continue to seek a more powerful model close to biological recognition to miRNA structure. In this report, we describe a novel miRNA prediction algorithm, known as FOMmiR, using a fixed-order Markov model based on the secondary structural pattern. For a training dataset containing 809 human pre-miRNAs and 6441 human pseudo-miRNA hairpins, the model's parameters were defined and evaluated. The results showed that FOMmiR reached 91% accuracy on the human dataset through 5-fold cross-validation. Moreover, for the independent test datasets, the FOMmiR presented an outstanding prediction in human and other species including vertebrates, Drosophila, worms and viruses, even plants, in contrast to the well-known algorithms and models. Especially, the FOMmiR was not only able to distinguish the miRNA precursors from the hairpins, but also locate the position and strand of the mature miRNA. Therefore, this study provides a new generation of miRNA prediction algorithm, which successfully realizes a full-function recognition of the mature miRNAs directly from the hairpin sequences. And it presents a new understanding of the biological recognition based on the strongest signal's location detected by FOMmiR, which might be closely associated with the enzyme cleavage mechanism during the miRNA maturation.

  12. Online Knowledge-Based Model for Big Data Topic Extraction

    Science.gov (United States)

    Khan, Muhammad Taimoor; Durrani, Mehr; Khalid, Shehzad; Aziz, Furqan

    2016-01-01

    Lifelong machine learning (LML) models learn with experience maintaining a knowledge-base, without user intervention. Unlike traditional single-domain models they can easily scale up to explore big data. The existing LML models have high data dependency, consume more resources, and do not support streaming data. This paper proposes online LML model (OAMC) to support streaming data with reduced data dependency. With engineering the knowledge-base and introducing new knowledge features the learning pattern of the model is improved for data arriving in pieces. OAMC improves accuracy as topic coherence by 7% for streaming data while reducing the processing cost to half. PMID:27195004

  13. Sequential Clustering based Facial Feature Extraction Method for Automatic Creation of Facial Models from Orthogonal Views

    CERN Document Server

    Ghahari, Alireza

    2009-01-01

    Multiview 3D face modeling has attracted increasing attention recently and has become one of the potential avenues in future video systems. We aim to make more reliable and robust automatic feature extraction and natural 3D feature construction from 2D features detected on a pair of frontal and profile view face images. We propose several heuristic algorithms to minimize possible errors introduced by prevalent nonperfect orthogonal condition and noncoherent luminance. In our approach, we first extract the 2D features that are visible to both cameras in both views. Then, we estimate the coordinates of the features in the hidden profile view based on the visible features extracted in the two orthogonal views. Finally, based on the coordinates of the extracted features, we deform a 3D generic model to perform the desired 3D clone modeling. Present study proves the scope of resulted facial models for practical applications like face recognition and facial animation.

  14. Extracting mining subsidence land from remote sensing images based on domain knowledge

    Institute of Scientific and Technical Information of China (English)

    WANG Xing-feng; WANG Yun-jia; HUANG Tai

    2008-01-01

    Extracting mining subsidence land from RS images is one of important research contents for environment monitoring in mining area. The accuracy of traditional extracting models based on spectral features is low. In order to extract subsidence land from RS images with high accuracy, some domain knowledge should be imported and new models should be proposed. This paper, in terms of the disadvantage of traditional extracting models, imports domain knowledge from practice and experience, converts semantic knowledge into digital information, and proposes a new model for the specific task. By selecting Luan mining area as study area, this new model is tested based on GIS and related knowledge. The result shows that the proposed method is more pre- cise than traditional methods and can satisfy the demands of land subsidence monitoring in mining area.

  15. Incorporation of garlic extract as antifungal agent in psyllium based edible coating for mandarin

    Directory of Open Access Journals (Sweden)

    Hafeez ur Rehman

    2015-07-01

    Full Text Available In present Research work, the mathanolic extract of garlic was incorporated in locally developed Psyllium based edible coating for its application on mandarin. Different concentrations of the extract were used in the coating and quality of the fruit was monitored during storage at room temperature. The results indicated that there was least change (increase in brix, weight loss, brix/acid ratio, pH and acidity of the fruit during storage studies.  The fungal contamination was effectively controlled due to incorporation of garlic extracts at a rate of 6-8%. On the basis of these results it was concluded that the garlic extracts can be used in psyllium based edible coating and it has antifungal significant antifungal potential but at relatively higher concentrations (>6%.

  16. Incorporation of garlic extract as antifungal agent in psyllium based edible coating for mandarin

    Directory of Open Access Journals (Sweden)

    Hafeez ur Rehman

    2015-08-01

    Full Text Available In present Research work, the mathanolic extract of garlic was incorporated in locally developed Psyllium based edible coating for its application on mandarin. Different concentrations of the extract were used in the coating and quality of the fruit was monitored during storage at room temperature. The results indicated that there was least change (increase in brix, weight loss, brix/acid ratio, pH and acidity of the fruit during storage studies.  The fungal contamination was effectively controlled due to incorporation of garlic extracts at a rate of 6-8%. On the basis of these results it was concluded that the garlic extracts can be used in psyllium based edible coating and it has antifungal significant antifungal potential but at relatively higher concentrations (>6%.

  17. Feature Extraction from 3D Point Cloud Data Based on Discrete Curves

    Directory of Open Access Journals (Sweden)

    Yi An

    2013-01-01

    Full Text Available Reliable feature extraction from 3D point cloud data is an important problem in many application domains, such as reverse engineering, object recognition, industrial inspection, and autonomous navigation. In this paper, a novel method is proposed for extracting the geometric features from 3D point cloud data based on discrete curves. We extract the discrete curves from 3D point cloud data and research the behaviors of chord lengths, angle variations, and principal curvatures at the geometric features in the discrete curves. Then, the corresponding similarity indicators are defined. Based on the similarity indicators, the geometric features can be extracted from the discrete curves, which are also the geometric features of 3D point cloud data. The threshold values of the similarity indicators are taken from [0,1], which characterize the relative relationship and make the threshold setting easier and more reasonable. The experimental results demonstrate that the proposed method is efficient and reliable.

  18. Diagonal Based Feature Extraction for Handwritten Alphabets Recognition System Using Neural Network

    Directory of Open Access Journals (Sweden)

    J.Pradeep

    2011-02-01

    Full Text Available An off-line handwritten alphabetical character recognition system using multilayer feed forward neuralnetwork is described in the paper. A new method, called, diagonal based feature extraction is introducedfor extracting the features of the handwritten alphabets. Fifty data sets, each containing 26 alphabetswritten by various people, are used for training the neural network and 570 different handwrittenalphabetical characters are used for testing. The proposed recognition system performs quite wellyielding higher levels of recognition accuracy compared to the systems employing the conventionalhorizontal and vertical methods of feature extraction. This system will be suitable for convertinghandwritten documents into structural text form and recognizing handwritten names.

  19. Diagonal Based Feature Extraction for Handwritten Alphabets Recognition System using Neural Network

    CERN Document Server

    Pradeep, J; Himavathi, S; 10.5121/ijcsit.2011.3103

    2011-01-01

    An off-line handwritten alphabetical character recognition system using multilayer feed forward neural network is described in the paper. A new method, called, diagonal based feature extraction is introduced for extracting the features of the handwritten alphabets. Fifty data sets, each containing 26 alphabets written by various people, are used for training the neural network and 570 different handwritten alphabetical characters are used for testing. The proposed recognition system performs quite well yielding higher levels of recognition accuracy compared to the systems employing the conventional horizontal and vertical methods of feature extraction. This system will be suitable for converting handwritten documents into structural text form and recognizing handwritten names.

  20. Increasing the Target Prediction Accuracy of MicroRNA Based on Combination of Prediction Algorithms

    Directory of Open Access Journals (Sweden)

    Mohammed Q. Shatnawi

    2016-06-01

    Full Text Available MicroRNA is an oligonucleotide that plays a role in the pathogenesis of several diseases (mentioning Cancer. It is a non-coding RNA that is involved in the control of gene expression through the binding and inhibition of mRNA. In this study, three algorithms were implemented in WEKA software using two testing modes to analyze five datasets of miRNA families. The data mining techniques are used to compare the interactions of miRNA-mRNA that it either belongs to the same gene-family or to different families, and to establish a biological scheme that explains how the biological parameters are involved or less involved in miRNA-mRNA prediction. The factors that were involved in the prediction process includs match, mismatch, bulge, loop, and score to represent the binding characteristics, while the position, 3’UTR length, and chromosomal location and chromosomal categorizations represent the characteristics of the target mRNA. These attributes can provide an empirical guidance for study of specific miRNA family to scan the whole human genome for novel targets. This research provides promising results that can be utilized for current and future research in this field.