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Sample records for dna microarray profiling

  1. Gene expression profiles in varicose veins using complementary DNA microarray.

    PubMed

    Lee, Seokjong; Lee, Wonchae; Choe, Yoonseok; Kim, Dowon; Na, Gunyeon; Im, Sanguk; Kim, Jinoh; Kim, Moonkyu; Kim, Jungchul; Cho, Joonyong

    2005-04-01

    There has been little information reported about the genetic event concerning the pathophysiology of varicose vein (VV). The purpose of this study was to examine the differentiation of gene expression in the wall of VV using complementary deoxyribonucleic acid (cDNA) microarrays. The study was performed with four pairs of VVs and control veins (CVs). cDNA specimens of VVs were prepared from the ribonucleic acid-isolated VVs of patients who underwent venous obliteration, using radiofrequency, as well as from CVs of those who underwent aortocoronary bypass grafting. Each set of VVs and CVs was hybridized with high-density microarray containing 3,063 human cDNAs. The finding of microarray hybridization were scanned, analyzed, and classified with the cluster program. Among 3,063 cDNA clones, 82 genes were up-regulated in VVs, and some of the up-regulated genes, which were detected by cDNA microarray, including transforming growth factor 3-induced gene (BIGH3), tubulin, lumican, actinin, collagen type I, versican, actin, and tropomyosin, belonged to extracelluar matrix molecules, cytoskeletal proteins, or myofibroblasts. Many up-regulated genes were found in Ws by applying cDNA microarray. These gene profiles suggested a pathway associated with fibrosis and that wound healing might be related to the pathophysiology of VVs.

  2. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    EPA Science Inventory

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
    Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  3. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    EPA Science Inventory

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
    Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  4. Fundamental Patterns Underlying Neurotoxicity Revealed by DNA Microarray Expression Profiling

    DTIC Science & Technology

    2004-09-01

    microarray analysis of the dopaminergic cell line, SN4741 , revealed induction of stress indices following MPP* treatment (Chun et al., 2001). To...response to a wide range of cellular stresses including oxidative insult of the nigral dopaminergic cell line SN4741 with hydrogen peroxide or MPP* (Salinas

  5. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

    PubMed

    Abiko, Yoshimitsu; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Tsushima, Katsumasa; Ohta, Mitsuhiro; Sasahara, Hiroshige

    2004-03-01

    Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

  6. Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

    PubMed Central

    Bono, Hidemasa; Yagi, Ken; Kasukawa, Takeya; Nikaido, Itoshi; Tominaga, Naoko; Miki, Rika; Mizuno, Yosuke; Tomaru, Yasuhiro; Goto, Hitoshi; Nitanda, Hiroyuki; Shimizu, Daisuke; Makino, Hirochika; Morita, Tomoyuki; Fujiyama, Junshin; Sakai, Takehito; Shimoji, Takashi; Hume, David A.; Hayashizaki, Yoshihide; Okazaki, Yasushi

    2003-01-01

    The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed. PMID:12819129

  7. Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

    PubMed Central

    Renn, Susan CP; Aubin-Horth, Nadia; Hofmann, Hans A

    2004-01-01

    Background Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits. Results Here we present a microarray constructed with ~4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (~65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time). Conclusion This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms. PMID:15238158

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

    PubMed Central

    Peano, Clelia; Bicciato, Silvio; Corti, Giorgio; Ferrari, Francesco; Rizzi, Ermanno; Bonnal, Raoul JP; Bordoni, Roberta; Albertini, Alberto; Bernardi, Luigi Rossi; Donadio, Stefano; De Bellis, Gianluca

    2007-01-01

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

  9. Differential gene expression profiling of vocal fold polyps and Reinke's edema by complementary DNA microarray.

    PubMed

    Duflo, Suzy M; Thibeault, Susan L; Li, Wenhua; Smith, Marshall E; Schade, Goetz; Hess, Markus M

    2006-09-01

    Our purpose was to determine whether complementary DNA (cDNA) microarray analysis (MA) can establish distinct gene expression profiles for 2 phenotypically similar vocal fold lesions: Reinke's edema (RE) and polyps. Established transcript profiles can provide insight into the molecular and cellular processes involved in these diseases. Eleven RE specimens and 17 polyps were analyzed with MA for 8,745 genes. Further MA profiling was attempted within each lesion group to identify molecular markers for reflux exposure and smoking. Prediction analysis was used to predict lesion classification for 2 unclassified samples. A real-time polymerase chain reaction was performed to corroborate MA transcript levels for selected significant genes. Sixty-five genes were found to differentiate RE and polyps (p = .0088). For RE, 19 genes were differentiated for reflux exposure (p = .016). No genes were found to differentiate smokers from nonsmokers. For polyps, no genes were found to differentiate for reflux (p = .16) and smoking (p = .565). Categorization of unclassified lesions was possible with a minimum of 13 genes. We demonstrate the feasibility of benign lesion classification based on MA. Microarray analysis is useful not only for improving diagnosis and classification of such lesions, but also for potentially generating prognostic indicators and targets for therapy.

  10. Gene expression profiling of in Moniezia expansa at different developmental proglottids using cDNA microarray.

    PubMed

    Bo, Xinwen; Zhao, Wenjuan; Zhang, Hui; Kang, Lichao; Wang, Xinhua

    2012-04-01

    Gene expression profiles of Moniezia expansa proglottids at varying developmental stages were analysed using cDNA microarray. A total of 4,056 spots, including full length and partial complementary DNAs that represent novel, known, and control genes, were studied. Results indicated an up-regulation of 55 genes in immature proglottids, 134 genes in mature proglottids and 103 genes in gravid proglottids were up-regulated, and a down-regulation of 7 genes in immature proglottids, 68 genes in mature proglottids and 78 genes in gravid proglottids compared to controls (scolex-neck proglottids). Many of these genes were identified as transcription factors and were involved in functions such as metabolism, transport, protein biosynthesis, apoptosis, cell differentiation, cell communication and nucleic acid binding. Expression level alterations in UBE2A, Cavβ, RAD51, DAZ, PKAc and 2 unknown genes were confirmed by real-time quantitative polymerase chain reaction (RT-PCR). The complete microarray data set has been deposited in the NCBI Gene Expression Omnibus, GEO Series accession number GSE13982. Results provide a gene expression profile at various development stages of M. expansa proglottids, which prove invaluable in understanding the pathogenesis of the tapeworm and studying the genes concerned with reproductive organ development.

  11. Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays.

    PubMed

    Sun, Xiaoli; Jia, Yu; Wei, Yuanyu; Liu, Shuai; Yue, Baohong

    2016-07-01

    Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53‑mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53‑independent NS pathway. NS expression was silenced using lentivirus‑mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse‑transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c‑Jun N‑terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54‑independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.

  12. Identification of conserved core xylem gene sets: conifer cDNA microarray development, transcript profiling and computational analyses.

    PubMed

    Pavy, Nathalie; Boyle, Brian; Nelson, Colleen; Paule, Charles; Giguère, Isabelle; Caron, Sébastien; Parsons, Lee S; Dallaire, Nancy; Bedon, Frank; Bérubé, Hugo; Cooke, Janice; Mackay, John

    2008-01-01

    One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage. A microarray of 10,400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process. White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants. Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

  13. Gene profile in the spleen under massive partial hepatectomy using complementary DNA microarray and pathway analysis.

    PubMed

    Arakawa, Yusuke; Shimada, Mitsuo; Utsunomiya, Tohru; Imura, Satoru; Morine, Yuji; Ikemoto, Tetsuya; Mori, Hiroki; Kanamoto, Mami; Iwahashi, Shuichi; Saito, Yu; Takasu, Chie

    2014-08-01

    In general, the spleen is one of the abdominal organs connected by the portal system, and a splenectomy improves hepatic functions in the settings of partial hepatectomy (Hx) for portal hypertensive cases or living donor liver transplantation with excessive portal vein flow. Those precise mechanisms remain still unclear; therefore, we investigated the DNA expression profile in the spleen after 90% Hx in rats using complementary DNA microarray and pathway analysis. Messenger RNAs (mRNAs) were prepared from three rat spleens at each time point (0, 3, and 6 h after 90% Hx). Using the gene chip, mRNA was hybridized to Affymetrix GeneChip Rat Genome 230 2.0 Array (Affymetrix®) and pathway analysis was done with Ingenuity Pathway Analysis (IPA®). We determined the 3-h or 6-h/0-h ratio to assess the influence of Hx, and cut-off values were set at more than 2.0-fold or less than 1/2 (0.5)-fold. Chemokine activity-related genes including Cxcl1 (GRO1) and Cxcl2 (MIP-2) related pathway were upregulated in the spleen. Also, immediate early response genes including early growth response-1 (EGR1), FBJ murine osteosarcoma (FOS) and activating transcription factor 3 (ATF3) related pathway were upregulated in the spleen. We concluded that in the spleen the expression of numerous inflammatory-related genes would occur after 90% Hx. The spleen could take a harmful role and provide a negative impact during post Hx phase due to the induction of chemokine and transcription factors including GRO1 and EGR1. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  14. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  15. Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays

    PubMed Central

    Nash, John HE; Findlay, Wendy A; Luebbert, Christian C; Mykytczuk, Oksana L; Foote, Simon J; Taboada, Eduardo N; Carrillo, Catherine D; Boyd, Jessica M; Colquhoun, Duncan J; Reith, Michael E; Brown, Laura L

    2006-01-01

    Background Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. Results Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. Conclusion We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research. PMID:16522207

  16. DNA microarray mediated transcriptional profiling of Nitrosomonas europaea in response to linear alkylbenzene sulfonates.

    PubMed

    Urakawa, Hidetoshi; Matsumoto, Junpei; Inaba, Kazuho; Tsuneda, Satoshi

    2008-05-01

    Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.

  17. DNA Microarrays in Herbal Drug Research

    PubMed Central

    Chavan, Preeti; Joshi, Kalpana; Patwardhan, Bhushan

    2006-01-01

    Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts. PMID:17173108

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

    PubMed Central

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

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

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

    PubMed

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

    2003-03-01

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

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

    PubMed

    Kwiatkowski, Przemysław; Wierzbicki, Piotr; Kmieć, Andrzej; Godlewski, Janusz

    2012-06-11

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

  1. Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

    PubMed Central

    Ahn, Woong Shick; Kim, Ko-Woon; Bae, Su Mi; Yoon, Joo Hee; Lee, Joon Mo; Namkoong, Sung Eun; Kim, Jin Hong; Kim, Chong Kook; Lee, Young Joo; Kim, Yong-Wan

    2003-01-01

    This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels. PMID:14748746

  2. Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

    PubMed

    Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

    2015-06-01

    White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

  3. Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity

    PubMed Central

    2010-01-01

    Background The European sea bass (Dicentrarchus labrax) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for D. labrax. Results A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Probe design was positively completed for 19,035 target clusters; the oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant. Conclusions The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon

  4. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    PubMed

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  5. Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray.

    PubMed

    De Backer, M D; Ilyina, T; Ma, X J; Vandoninck, S; Luyten, W H; Vanden Bossche, H

    2001-06-01

    The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600 Candida albicans gene transcript levels, representing the entire genome, upon treatment of cells with 10 microM itraconazole revealed that 296 genes were responsive. For 116 genes transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation of ERG genes in response to azole treatment was observed. ERG11 and ERG5 were found to be upregulated approximately 12-fold. In addition, a significant upregulation was observed for ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25, ERG2, IDII, HMGS, NCP1, and FEN2, all of which are genes known to be involved in ergosterol biosynthesis. The effects of itraconazole on a wide variety of known metabolic processes are discussed. As over 140 proteins with unknown function were responsive to itraconazole, our analysis might provide-in combination with phenotypic data-first hints of their potential function. The present report is the first to describe the application of DNA chip technology to study the response of a major human fungal pathogen to drug treatment.

  6. Genomic Profiling of the Response of Candida albicans to Itraconazole Treatment Using a DNA Microarray

    PubMed Central

    De Backer, Marianne D.; Ilyina, Tatiana; Ma, Xiao-Jun; Vandoninck, Sandy; Luyten, Walter H. M. L.; Vanden Bossche, Hugo

    2001-01-01

    The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600 Candida albicans gene transcript levels, representing the entire genome, upon treatment of cells with 10 μM itraconazole revealed that 296 genes were responsive. For 116 genes transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation of ERG genes in response to azole treatment was observed. ERG11 and ERG5 were found to be upregulated approximately 12-fold. In addition, a significant upregulation was observed for ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25, ERG2, IDII, HMGS, NCP1, and FEN2, all of which are genes known to be involved in ergosterol biosynthesis. The effects of itraconazole on a wide variety of known metabolic processes are discussed. As over 140 proteins with unknown function were responsive to itraconazole, our analysis might provide—in combination with phenotypic data—first hints of their potential function. The present report is the first to describe the application of DNA chip technology to study the response of a major human fungal pathogen to drug treatment. PMID:11353609

  7. DNA Microarray-Based Diagnostics.

    PubMed

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

    2016-01-01

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

  8. Progress in the application of DNA microarrays.

    PubMed Central

    Lobenhofer, E K; Bushel, P R; Afshari, C A; Hamadeh, H K

    2001-01-01

    Microarray technology has been applied to a variety of different fields to address fundamental research questions. The use of microarrays, or DNA chips, to study the gene expression profiles of biologic samples began in 1995. Since that time, the fundamental concepts behind the chip, the technology required for making and using these chips, and the multitude of statistical tools for analyzing the data have been extensively reviewed. For this reason, the focus of this review will be not on the technology itself but on the application of microarrays as a research tool and the future challenges of the field. PMID:11673116

  9. Analysis of DNA microarray expression data.

    PubMed

    Simon, Richard

    2009-06-01

    DNA microarrays are powerful tools for studying biological mechanisms and for developing prognostic and predictive classifiers for identifying the patients who require treatment and are best candidates for specific treatments. Because microarrays produce so much data from each specimen, they offer great opportunities for discovery and great dangers or producing misleading claims. Microarray based studies require clear objectives for selecting cases and appropriate analysis methods. Effective analysis of microarray data, where the number of measured variables is orders of magnitude greater than the number of cases, requires specialized statistical methods which have recently been developed. Recent literature reviews indicate that serious problems of analysis exist a substantial proportion of publications. This manuscript attempts to provide a non-technical summary of the key principles of statistical design and analysis for studies that utilize microarray expression profiling.

  10. DNA Microarray Technology

    SciTech Connect

    WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

    2002-01-01

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

  11. Application of DNA microarrays in occupational health research.

    PubMed

    Koizumi, Shinji

    2004-01-01

    The profiling of gene expression patterns with DNA microarrays is recently being widely used not only in basic molecular biological studies but also in the practical fields. In clinical application, for example, this technique is expected to be quite useful in making a correct diagnosis. In the pharmacological area, the microarray analysis can be applied to drug discovery and individualized drug treatment. Although not so popular as these examples, DNA microarrays could also be a powerful tool in studies relevant to occupational health. This review will describe the outline of gene expression profiling with DNA microarrays and prospects in occupational health research.

  12. Analysis of mechanisms of TPA induced differentiation in HL-60 cells by expression profiling with DNA microarray.

    SciTech Connect

    Zheng, X.; Ravatn, R.; Lin, Y.; Shih, W.-C.; Rabson, A.; Strair, R.; Huberman, E.; Conney, A.; Chin, K.-V.; Biochip Technology Center

    2002-10-15

    The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of differentiation in human promyelocytic leukemia cells. Recently, TPA has been successfully administered to patients with myelocytic leukemia and has produced therapeutic effects that led to temporary remission. These studies demonstrated the potential efficacy of TPA in cancer chemotherapy. We now seek to understand the biological effects and molecular mechanisms of differentiation in response to TPA treatment in leukemia cells by expression profiling using DNA microarray. Our results show distinct temporal and coordinated gene changes that are consistent with differentiation and activation of multiple biochemical pathways in HL-60 cells exposed to TPA. Alterations of gene expression in HL-60 cells include various transcription factors, cytokines and protein markers that are consistent with the induction of differentiation elicited by TPA. These temporal patterns of gene expression were abolished or greatly diminished in an HL-60 derived TPA- resistant variant cell line (HL-525), thus revealing transcriptional and consequential biochemical changes that may be required for TPA-induced differentiation. In addition, certain genes were upregulated by TPA in TPA-resistant HL-525 cells but not in TPA-sensitive HL-60 cells suggesting that these genes may play a role in the resistant pheno type. These patterns of gene expression may be important for predicting response to TPA.

  13. DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

    PubMed

    Trio, Phoebe Zapanta; Kawahara, Atsuyoshi; Tanigawa, Shunsuke; Sakao, Kozue; Hou, De-Xing

    2017-01-01

    6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

  14. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  15. DNA microarray integromics analysis platform.

    PubMed

    Waller, Tomasz; Gubała, Tomasz; Sarapata, Krzysztof; Piwowar, Monika; Jurkowski, Wiktor

    2015-01-01

    The study of interactions between molecules belonging to different biochemical families (such as lipids and nucleic acids) requires specialized data analysis methods. This article describes the DNA Microarray Integromics Analysis Platform, a unique web application that focuses on computational integration and analysis of "multi-omics" data. Our tool supports a range of complex analyses, including - among others - low- and high-level analyses of DNA microarray data, integrated analysis of transcriptomics and lipidomics data and the ability to infer miRNA-mRNA interactions. We demonstrate the characteristics and benefits of the DNA Microarray Integromics Analysis Platform using two different test cases. The first test case involves the analysis of the nutrimouse dataset, which contains measurements of the expression of genes involved in nutritional problems and the concentrations of hepatic fatty acids. The second test case involves the analysis of miRNA-mRNA interactions in polysaccharide-stimulated human dermal fibroblasts infected with porcine endogenous retroviruses. The DNA Microarray Integromics Analysis Platform is a web-based graphical user interface for "multi-omics" data management and analysis. Its intuitive nature and wide range of available workflows make it an effective tool for molecular biology research. The platform is hosted at https://lifescience.plgrid.pl/.

  16. Strategy for the design of custom cDNA microarrays.

    PubMed

    Lorenz, Matthias G O; Cortes, Lizette M; Lorenz, Juergen J; Liu, Edison T

    2003-06-01

    DNA microarrays are valuable but expensive tools for expression profiling of cells, tissues, and organs. The design of custom microarrays leads to cost reduction without necessarily compromising their biological value. Here we present a strategy for designing custom cDNA microarrays and constructed a microarray for mouse immunology research (ImmunoChip). The strategy used interrogates expressed sequence tag databases available in the public domain but overcomes many of the problems encountered. Immunologically relevant clusters were selected based on the expression of expressed sequence tags in relevant libraries. Selected clusters were organized in modules, and the best representative clones were identified. When tested, this microarray was found to have minimal clone identity errors or phage contamination and identified molecular signatures of lymphoid cell lines. Our proposed design of custom microarrays avoids probe redundancy, allows the organization of the chip to optimize chip production, and reduces microarray production costs. The strategy described is also useful for the design of oligonucleotide microarrays.

  17. DNA microarray technology in dermatology.

    PubMed

    Kunz, Manfred

    2008-03-01

    In recent years, DNA microarray technology has been used for the analysis of gene expression patterns in a variety of skin diseases, including malignant melanoma, psoriasis, lupus erythematosus, and systemic sclerosis. Many of the studies described herein confirmed earlier results on individual genes or functional groups of genes. However, a plethora of new candidate genes, gene patterns, and regulatory pathways have been identified. Major progresses were reached by the identification of a prognostic gene pattern in malignant melanoma, an immune signaling cluster in psoriasis, and a so-called interferon signature in systemic lupus erythematosus. In future, interference with genes or regulatory pathways with the use of different RNA interference technologies or targeted therapy may not only underscore the functional significance of microarray data but also may open interesting therapeutic perspectives. Large-scale gene expression analyses may also help to design more individualized treatment approaches of cutaneous diseases.

  18. Monitoring of gene expression profiles and isolation of candidate genes involved in pollination and fertilization in rice ( Oryza sativa L.) with a 10K cDNA microarray.

    PubMed

    Lan, Lefu; Chen, Wei; Lai, Ying; Suo, Jinfeng; Kong, Zhaosheng; Li, Can; Lu, Ying; Zhang, Yujun; Zhao, Xiangyu; Zhang, Xiansheng; Zhang, Yansheng; Han, Bin; Cheng, Jing; Xue, Yongbiao

    2004-03-01

    To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.

  19. Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology.

    PubMed

    Sarropoulou, Elena; Kotoulas, Georgios; Power, Deborah M; Geisler, Robert

    2005-10-17

    Large-scale gene expression studies were performed for one of the main European aquaculture species, the gilthead sea bream Sparus auratus L. For this purpose, a cDNA microarray containing 10,176 clones from a cDNA library of mixed embryonic and larval stages was constructed. In addition to its importance for aquaculture, the taxonomic position and the relatively small genome size of sea bream makes it a prospective model for evolutionary biology and comparative genomics. However, so far, no large-scale analysis of gene expression exists for this species. In the present study, gene expression was analyzed in gilthead sea bream during early development, a significant period in the determination of quantitative traits and therefore of considerable interest for aquaculture. Synexpression groups expressed primarily early and late in development were determined and were composed of both known and novel genes. Furthermore, it was possible to identify stress response genes induced by cortisol injections using the cDNA microarray generated. The creation of gene expression profiles for sea bream by microarray hybridization will accelerate identification of candidate genes involved in multifactorial traits and certain regulatory pathways and will also contribute to a better understanding of the genetic background of fish physiology, which may help to improve aquaculture practices.

  20. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

    PubMed

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

  1. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and {gamma}-rays

    SciTech Connect

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep . E-mail: rakwal-68@aist.go.jp; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma ({gamma})-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and {gamma}-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and {gamma}-rays). Similarly, for X- and {gamma}-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and {gamma}-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-a-vis their energy levels.

  2. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  3. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  4. Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray.

    PubMed

    Nie, Zhiyi; Kang, Guijuan; Duan, Cuifang; Li, Yu; Dai, Longjun; Zeng, Rizhong

    2016-01-01

    Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ -2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis.

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

    PubMed Central

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

    2015-01-01

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

  6. Use of Genomic DNA as A Reference in DNA Microarrays

    SciTech Connect

    Yang, Yunfeng

    2009-01-01

    DNA microarray has become a mainstream technology to explore gene expression profiles, identify novel genes involved in a biological process of interest and predict their function, and determine biomarkers that are relevant to a given phenotype or disease. Typical two-channel microarray studies use an experimental design called the complementary DNA (cDNA) reference method, in which samples from test and control conditions are compared directly on a microarray slide. A substantial limitation of this strategy is that it is nearly impossible to compare data between experiments because the reference sample composition is subjected to changes at the level of experimental design and thereby not consistent from one experiment to another. Using genomic DNA as common reference will effectively overcome this limitation. This chapter describes detailed methods to prepare genomic DNA of high quality, label with fluorescent dye, co-hybridize with cDNA samples, and the subsequent data analyses. In addition, notes are provided to help the readers to obtain optimal results using the procedure.

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

    PubMed Central

    Datson, Nicole A; Morsink, Maarten C; Atanasova, Srebrena; Armstrong, Victor W; Zischler, Hans; Schlumbohm, Christina; Dutilh, Bas E; Huynen, Martijn A; Waegele, Brigitte; Ruepp, Andreas; de Kloet, E Ronald; Fuchs, Eberhard

    2007-01-01

    Background The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native to eastern Brazil, is becoming increasingly used as a non-human primate model in biomedical research, drug development and safety assessment. In contrast to the growing interest for the marmoset as an animal model, the molecular tools for genetic analysis are extremely limited. Results Here we report the development of the first marmoset-specific oligonucleotide microarray (EUMAMA) containing probe sets targeting 1541 different marmoset transcripts expressed in hippocampus. These 1541 transcripts represent a wide variety of different functional gene classes. Hybridisation of the marmoset microarray with labelled RNA from hippocampus, cortex and a panel of 7 different peripheral tissues resulted in high detection rates of 85% in the neuronal tissues and on average 70% in the non-neuronal tissues. The expression profiles of the 2 neuronal tissues, hippocampus and cortex, were highly similar, as indicated by a correlation coefficient of 0.96. Several transcripts with a tissue-specific pattern of expression were identified. Besides the marmoset microarray we have generated 3215 ESTs derived from marmoset hippocampus, which have been annotated and submitted to GenBank [GenBank: EF214838 – EF215447, EH380242 – EH382846]. Conclusion We have generated the first marmoset-specific DNA microarray and demonstrated its use to characterise large-scale gene expression profiles of hippocampus but also of other neuronal and non-neuronal tissues. In addition, we have generated a large collection of ESTs of marmoset origin, which are now available in the public domain. These new tools will facilitate molecular genetic research into this non-human primate animal model. PMID:17592630

  8. Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling in Magnaporthe oryzae

    PubMed Central

    Wáng, Ying; Tan, Qi; Gao, Ying Nv; Li, Yan

    2017-01-01

    High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity in Magnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity in M. oryzae identified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected. PMID:28286772

  9. Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling in Magnaporthe oryzae.

    PubMed

    Wang, Ying; Wáng, Ying; Tan, Qi; Gao, Ying Nv; Li, Yan; Bao, Da Peng

    2017-01-01

    High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity in Magnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity in M. oryzae identified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.

  10. Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray

    PubMed Central

    Nie, Zhiyi; Kang, Guijuan; Duan, Cuifang; Li, Yu; Dai, Longjun; Zeng, Rizhong

    2016-01-01

    Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ –2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis. PMID:26985821

  11. cDNA microarray assessment of early gene expression profiles in Escherichia coli cells exposed to a mixture of heavy metals.

    PubMed

    Gómez-Sagasti, María T; Becerril, José M; Martín, Iker; Epelde, Lur; Garbisu, Carlos

    2014-08-01

    Many contaminated sites are characterized by the presence of different metals, thus increasing the complexity of toxic responses in exposed organisms. Within toxicogenomics, transcriptomics can be approached through the use of microarrays aimed at producing a genetic fingerprint for the response of model organisms to the presence of chemicals. We studied temporal changes in the early gene expression profiles of Escherichia coli cells exposed to three metal doses of a polymetallic solution over three exposure times, through the application of cDNA microarray technology. In the absence of metals, many genes belonging to a variety of cellular functions were up- and down-regulated over time. At the lowest metal dose, an activation of metal-specific transporters (Cus and ZraP proteins) and a mobilization of glutathione transporters involved in metal sequestration and trafficking was observed over time; this metal dose resulted in the generation of ROS capable of stimulating the transcription of Mn-superoxide dismutase, the assembly of Fe-S clusters and the synthesis of cysteine. At the intermediate dose, an overexpression of ROS scavengers (AhpF, KatG, and YaaA) and heat shock proteins (ClpP, HslV, DnaK, and IbpAB) was observed. Finally, at the highest dose, E. coli cells showed a repression of genes related with DNA mutation correctors (MutY glycopeptidases).

  12. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manufacturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthesized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microarrays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  13. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manu facturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthe-sized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microar-rays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-03

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

  16. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

    PubMed

    Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav

    2003-12-01

    Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

  17. Comprehensive Expression Profiling of Rice Grain Filling-Related Genes under High Temperature Using DNA Microarray[OA

    PubMed Central

    Yamakawa, Hiromoto; Hirose, Tatsuro; Kuroda, Masaharu; Yamaguchi, Takeshi

    2007-01-01

    To elucidate the effect of high temperature on grain-filling metabolism, developing rice (Oryza sativa) ‘Nipponbare’ caryopses were exposed to high temperature (33°C/28°C) or control temperature (25°C/20°C) during the milky stage. Comprehensive gene screening by a 22-K DNA microarray and differential hybridization, followed by expression analysis by semiquantitative reverse transcription-PCR, revealed that several starch synthesis-related genes, such as granule-bound starch synthase I (GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate dikinase gene were down-regulated by high temperature, whereas those for starch-consuming α-amylases and heat shock proteins were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with the diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted in the occurrence of grains with various degrees of chalky appearance and decreased weight. Among them, severely chalky grains contained amylopectin enriched particularly with long chains compared to slightly chalky grains, suggesting that such alterations of amylopectin structure might be involved in grain chalkiness. However, among high temperature-tolerant and sensitive cultivars, alterations of neither amylopectin chain-length distribution nor amylose content were correlated to the degree of grain chalkiness, but rather seemed to be correlated to grain weight decrease, implying different underlying mechanisms for the varietal difference in grain chalkiness. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are

  18. DNA microarray-mediated transcriptional profiling of avian pathogenic Escherichia coli O2 strain E058 during its infection of chicken.

    PubMed

    Gao, Qingqing; Xia, Le; Liu, Juanhua; Wang, Xiaobo; Gao, Song; Liu, Xiufan

    2016-11-01

    Avian pathogenic Escherichia coli (APEC) cause typical extraintestinal infections in poultry, including acute fatal septicemia, subacute pericarditis, and airsacculitis. These bacteria most often infect chickens, turkeys, ducks, and other avian species, and therefore pose a significant economic burden on the poultry industry worldwide. Few studies have analyzed the genome-wide transcriptional profile of APEC during infection in vivo. In this study, we examined the genome-wide transcriptional response of APEC O2 strain E058 in an in vivo chicken infection model to better understand the factors necessary for APEC colonization, growth, and survival in vivo. An Affymetrix multigenome DNA microarray, which contains most of the genomic open reading frames of E. coli K-12 strain MG1655, uropathogenic E. coli strain CFT073, and E. coli O157:H7 strain EDL 933, was used to profile the gene expression in APEC E058. We identified the in vivo transcriptional response of APEC E058 bacteria collected directly from the blood of infected chickens. Significant differences in expression levels were detected between the in vivo expression profile and the in vitro expression profile in LB medium. The genes highly expressed during infection were involved in metabolism, iron acquisition or transport, virulence, response to stress, and biological regulation. The reliability of the microarray data was confirmed by performing quantitative real-time PCR on 12 representative genes. Moreover, several significantly upregulated genes, including yjiY, sodA, phoB and spy, were selected to study their role in APEC pathogenesis. The data will help to better understand the mechanisms of APEC pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Application of a cDNA microarray for profiling the gene expression of Echinococcus granulosus protoscoleces treated with albendazole and artemisinin.

    PubMed

    Lü, Guodong; Zhang, Wenbao; Wang, Jianhua; Xiao, Yunfeng; Zhao, Jun; Zhao, Jianqin; Sun, Yimin; Zhang, Chuanshan; Wang, Junhua; Lin, Renyong; Liu, Hui; Zhang, Fuchun; Wen, Hao

    2014-12-01

    Cystic echinoccocosis (CE) is a neglected zoonosis that is caused by the dog-tapeworm Echinococcus granulosus. The disease is endemic worldwide. There is an urgent need for searching effective drug for the treatment of the disease. In this study, we sequenced a cDNA library constructed using RNA isolated from oncospheres, protoscoleces, cyst membrane and adult worms of E. granulosus. A total of 9065 non-redundant or unique sequences were obtained and spotted on chips as uniEST probes to profile the gene expression in protoscoleces of E. granulosus treated with the anthelmintic drugs albendazole and artemisinin, respectively. The results showed that 7 genes were up-regulated and 38 genes were down-regulated in the protoscoleces treated with albendazole. Gene analysis showed that these genes are responsible for energy metabolism, cell cycle and assembly of cell structure. We also identified 100 genes up-regulated and 6 genes down-regulated in the protoscoleces treated with artemisinin. These genes play roles in the transduction of environmental signals, and metabolism. Albendazole appeared its drug efficacy in damaging cell structure, while artemisinin was observed to increase the formation of the heterochromatin in protoscolex cells. Our results highlight the utility of using cDNA microarray methods to detect gene expression profiles of E. granulosus and, in particular, to understand the pharmacologic mechanism of anti-echinococcosis drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages.

    PubMed

    Li, Jiangning; Pritchard, David K; Wang, Xi; Park, David R; Bumgarner, Roger E; Schwartz, Stephen M; Liles, W Conrad

    2007-01-01

    We report the systematic use of large-scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro-differentiated, M-CSF-induced MONO-derived macrophages (MAC) and primary human alveolar MAC (AM), obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large-scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition, large differences were observed in the gene expression profiles of the two MAC types. Specifically, 21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO, and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes.

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

    SciTech Connect

    Gregory Stephanopoulos

    2004-07-31

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

  2. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The…

  3. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The…

  4. Identifying Fishes through DNA Barcodes and Microarrays

    PubMed Central

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

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

  5. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  6. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  7. Profiling In Situ Microbial Community Structure with an Amplification Microarray

    PubMed Central

    Knickerbocker, Christopher; Bryant, Lexi; Golova, Julia; Wiles, Cory; Williams, Kenneth H.; Peacock, Aaron D.; Long, Philip E.

    2013-01-01

    The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO3−) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO3, but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications. PMID:23160129

  8. Hybridization and Selective Release of DNA Microarrays

    SciTech Connect

    Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J; Benett, W; Mukerjee, E; Dzenitis, J; Wheeler, E K

    2011-11-29

    DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to

  9. DNA microarrays: an introduction to the technology.

    PubMed

    Bilitewski, Ursula

    2009-01-01

    DNA microarrays allow the comprehensive genetic analysis of an organism or a sample. They are based on probes, which are immobilized in an ordered two-dimensional pattern on substrates, such as nylon membranes or glass slides. Probes are either spotted cDNAs or oligonucleotides and are designed to be specific for an organism, a gene, a genetic variant (mutation or polymorphism), or intergenic regions. Thus, they can be used for example for genotyping, expression analysis, or studies of protein-DNA interactions, and in the biomedical field they allow the detection of pathogens, antibiotic resistances, gene mutations and polymorphisms, and pathogenic states and can guide therapy. Microarrays, which cover the whole genome of an organism, are as well available as those which are focussed on genes related to a certain diagnostic application.

  10. Giant Magnetoresistive Sensors for DNA Microarray

    PubMed Central

    Xu, Liang; Yu, Heng; Han, Shu-Jen; Osterfeld, Sebastian; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2009-01-01

    Giant magnetoresistive (GMR) sensors are developed for a DNA microarray. Compared with the conventional fluorescent sensors, GMR sensors are cheaper, more sensitive, can generate fully electronic signals, and can be easily integrated with electronics and microfluidics. The GMR sensor used in this work has a bottom spin valve structure with an MR ratio of 12%. The single-strand target DNA detected has a length of 20 bases. Assays with DNA concentrations down to 10 pM were performed, with a dynamic range of 3 logs. A double modulation technique was used in signal detection to reduce the 1/f noise in the sensor while circumventing electromagnetic interference. The logarithmic relationship between the magnetic signal and the target DNA concentration can be described by the Temkin isotherm. Furthermore, GMR sensors integrated with microfluidics has great potential of improving the sensitivity to 1 pM or below, and the total assay time can be reduced to less than 1 hour. PMID:20824116

  11. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  12. Overview of DNA microarrays: types, applications, and their future.

    PubMed

    Bumgarner, Roger

    2013-01-01

    This unit provides an overview of DNA microarrays. Microarrays are a technology in which thousands of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. This overview first discusses the history of microarrays and the antecedent technologies that led to their development. This is followed by discussion of the methods of manufacture of microarrays and the most common biological applications. The unit ends with a brief description of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.

  13. Electrostatic readout of DNA microarrays with charged microspheres

    PubMed Central

    Clack, Nathan G; Salaita, Khalid; Groves, Jay T

    2014-01-01

    DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis1–3. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care4,5. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-μm lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays. PMID:18587384

  14. Electrostatic readout of DNA microarrays with charged microspheres

    SciTech Connect

    Clack, Nathan G.; Salaita, Khalid; Groves, Jay T.

    2008-06-29

    DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. In this paper, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-μm lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Lastly, because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.

  15. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  16. DNA microarray-based PCR ribotyping of Clostridium difficile.

    PubMed

    Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2015-02-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.

  17. E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.

    PubMed

    Urisman, Anatoly; Fischer, Kael F; Chiu, Charles Y; Kistler, Amy L; Beck, Shoshannah; Wang, David; DeRisi, Joseph L

    2005-01-01

    DNA microarrays may be used to identify microbial species present in environmental and clinical samples. However, automated tools for reliable species identification based on observed microarray hybridization patterns are lacking. We present an algorithm, E-Predict, for microarray-based species identification. E-Predict compares observed hybridization patterns with theoretical energy profiles representing different species. We demonstrate the application of the algorithm to viral detection in a set of clinical samples and discuss its relevance to other metagenomic applications.

  18. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray.

    PubMed

    Qiu, Wen-Ming; Zhu, An-Dan; Wang, Yao; Chai, Li-Jun; Ge, Xiao-Xia; Deng, Xiu-Xin; Guo, Wen-Wu

    2012-08-16

    Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated. Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.

  19. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray

    PubMed Central

    2012-01-01

    Background Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. Results An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated. Conclusion Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future. PMID:22897898

  20. DNA microarray-based mutation discovery and genotyping.

    PubMed

    Gresham, David

    2011-01-01

    DNA microarrays provide an efficient means of identifying single-nucleotide polymorphisms (SNPs) in DNA samples and characterizing their frequencies in individual and mixed samples. We have studied the parameters that determine the sensitivity of DNA probes to SNPs and found that the melting temperature (T (m)) of the probe is the primary determinant of probe sensitivity. An isothermal-melting temperature DNA microarray design, in which the T (m) of all probes is tightly distributed, can be implemented by varying the length of DNA probes within a single DNA microarray. I describe guidelines for designing isothermal-melting temperature DNA microarrays and protocols for labeling and hybridizing DNA samples to DNA microarrays for SNP discovery, genotyping, and quantitative determination of allele frequencies in mixed samples.

  1. Gene expression profiling of mouse embryos with microarrays

    PubMed Central

    Sharov, Alexei A.; Piao, Yulan; Ko, Minoru S. H.

    2011-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. PMID:20699157

  2. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  3. Integrated microfluidic biochips for DNA microarray analysis.

    PubMed

    Liu, Robin Hui; Dill, Kilian; Fuji, H Sho; McShea, Andy

    2006-03-01

    A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

  4. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray

    PubMed Central

    Zhou, Gao-Feng; Liu, Yong-Zhong; Sheng, Ou; Wei, Qing-Jiang; Yang, Cheng-Quan; Peng, Shu-Ang

    2015-01-01

    Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus. PMID:25674093

  5. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

    PubMed

    Zhou, Gao-Feng; Liu, Yong-Zhong; Sheng, Ou; Wei, Qing-Jiang; Yang, Cheng-Quan; Peng, Shu-Ang

    2014-01-01

    Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

  6. Modeling background intensity in DNA microarrays

    NASA Astrophysics Data System (ADS)

    Kroll, K. M.; Barkema, G. T.; Carlon, E.

    2008-06-01

    DNA microarrays are devices that are able, in principle, to detect and quantify the presence of specific nucleic acid sequences in complex biological mixtures. The measurement consists in detecting fluorescence signals from several spots on the microarray surface onto which different probe sequences are grafted. One of the problems of the data analysis is that the signal contains a noisy background component due to nonspecific binding. We present a physical model for background estimation in Affymetrix Genechips. It combines two different approaches. The first is based on the sequence composition, specifically its sequence-dependent hybridization affinity. The second is based on the strong correlation of intensities from locations which are the physical neighbors of a specific spot on the chip. Both effects are incorporated in a background estimator which contains 24 free parameters, fixed by minimization on a training data set. In all data analyzed the sequence-specific parameters, obtained by minimization, are found to strongly correlate with empirically determined stacking free energies for RNA-DNA hybridization in solution. Moreover, there is an overall agreement with experimental background data and we show that the physics-based model that we propose performs on average better than purely statistical approaches for background calculations. The model thus provides an interesting alternative method for background subtraction schemes in Affymetrix Genechips.

  7. Independent component analysis of Alzheimer's DNA microarray gene expression data

    PubMed Central

    Kong, Wei; Mou, Xiaoyang; Liu, Qingzhong; Chen, Zhongxue; Vanderburg, Charles R; Rogers, Jack T; Huang, Xudong

    2009-01-01

    Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA) and independent component analysis (ICA) have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD) hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In comparison to the PCA and support

  8. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  9. DNA microarray for detection of gastrointestinal viruses.

    PubMed

    Martínez, Miguel A; Soto-Del Río, María de Los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y; Greninger, Alexander L; Contreras, Juan Francisco; López, Susana; Arias, Carlos F; Isa, Pavel

    2015-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  10. Microarrays (DNA chips) for the classroom laboratory.

    PubMed

    Barnard, Betsy; Sussman, Michael; Bondurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-09-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The primary adaptation is the use of a simulated cDNA target. The low density DNA array we discuss here was used to demonstrate differential expression of several Arabidopsis thaliana genes related to photosynthesis and photomorphogenesis. The methods we present here can be used with any biological organism whose sequence is known. Furthermore, these methods can be adapted to exhibit a variety of differential gene expression patterns under different experimental conditions. The materials and tools we discuss have been applied in classrooms at West High School in Madison, WI. We have also shared these materials with high school teachers attending professional development courses at the University of Wisconsin-Madison.

  11. Uses of Dendrimers for DNA Microarrays

    PubMed Central

    Caminade, Anne-Marie; Padié, Clément; Laurent, Régis; Maraval, Alexandrine; Majoral, Jean-Pierre

    2006-01-01

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

  12. Microarray analysis of DNA replication timing.

    PubMed

    Karnani, Neerja; Taylor, Christopher M; Dutta, Anindya

    2009-01-01

    Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic or asynchronous replication, which otherwise are difficult to detect at a genome-wide scale by existing methods. The continuous TR50 (time of completion of 50% replication) maps of replication across chromosomal segments identify regions that undergo acute transitions in replication timing. These transition zones can play a significant role in identifying insulators that separate chromosomal domains with different chromatin modifications.

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

    PubMed Central

    2010-01-01

    Background The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to 252Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes. Methods HeLa cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR. Results The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to 252Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated. Conclusions Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these "radioresistance" genes will lead to new therapeutic targets for cervical cancer. PMID:20184742

  14. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.

  15. Tissue microarray profiling in human heart failure.

    PubMed

    Lal, Sean; Nguyen, Lisa; Tezone, Rhenan; Ponten, Fredrik; Odeberg, Jacob; Li, Amy; Dos Remedios, Cristobal

    2016-09-01

    Tissue MicroArrays (TMAs) are a versatile tool for high-throughput protein screening, allowing qualitative analysis of a large number of samples on a single slide. We have developed a customizable TMA system that uniquely utilizes cryopreserved human cardiac samples from both heart failure and donor patients to produce formalin-fixed paraffin-embedded sections. Confirmatory upstream or downstream molecular studies can then be performed on the same (biobanked) cryopreserved tissue. In a pilot study, we applied our TMAs to screen for the expression of four-and-a-half LIM-domain 2 (FHL2), a member of the four-and-a-half LIM family. This protein has been implicated in the pathogenesis of heart failure in a variety of animal models. While FHL2 is abundant in the heart, not much is known about its expression in human heart failure. For this purpose, we generated an affinity-purified rabbit polyclonal anti-human FHL2 antibody. Our TMAs allowed high-throughput profiling of FHL2 protein using qualitative and semiquantitative immunohistochemistry that proved complementary to Western blot analysis. We demonstrated a significant relative reduction in FHL2 protein expression across different forms of human heart failure. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Alteration of Gene Expression Profile in Kidney of Spontaneously Hypertensive Rats Treated with Protein Hydrolysate of Blue Mussel (Mytilus edulis) by DNA Microarray Analysis

    PubMed Central

    Feng, Junli; Dai, Zhiyuan; Zhang, Yanping; Meng, Lu; Ye, Jian; Ma, Xuting

    2015-01-01

    Marine organisms are rich sources of bioactive components, which are often reported to have antihypertensive effects. However, the underlying mechanisms have yet to be fully identified. The aim of this study was to investigate the antihypertensive effect of enzymatic hydrolysis of blue mussel protein (HBMP) in rats. Peptides with in vitro ACE inhibitory activity were purified from HBMP by ultrafiltration, gel filtration chromatography and reversed-phase high performance liquid chromatography. And the amino acid sequences of isolated peptides were estimated to be Val-Trp, Leu-Gly-Trp, and Met-Val-Trp-Thr. To study its in vivo action, spontaneously hypertensive rats (SHRs) were orally administration with high- or low-dose of HBMP for 28 days. Major components of the renin-angiotensin (RAS) system in serum of SHRs from different groups were analyzed, and gene expression profiling were performed in the kidney of SHRs, using the Whole Rat Genome Oligonucleotide Microarray. Results indicated although genes involved in RAS system were not significantly altered, those related to blood coagulation system, cytokine and growth factor, and fatty acids metabolism were remarkablely changed. Several genes which were seldom reported to be implicated in pathogenesis of hypertension also showed significant expression alterations after oral administration of HBMP. These data provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of HBMP, and will contribute towards increased value-added utilization of blue mussel protein. PMID:26517713

  17. Analysis of Mycobacterium leprae gene expression using DNA microarray.

    PubMed

    Akama, Takeshi; Tanigawa, Kazunari; Kawashima, Akira; Wu, Huhehasi; Ishii, Norihisa; Suzuki, Koichi

    2010-10-01

    Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

  18. Application of DNA microarray technology to gerontological studies.

    PubMed

    Masuda, Kiyoshi; Kuwano, Yuki; Nishida, Kensei; Rokutan, Kazuhito

    2013-01-01

    Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.

  19. Analysis of Microarray and RNA-seq Expression Profiling Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Gene expression profiling refers to the simultaneous measurement of the expression levels of a large number of genes (often all genes in a genome), typically in multiple experiments spanning a variety of cell types, treatments, or environmental conditions. Expression profiling is accomplished by assaying mRNA levels with microarrays or next-generation sequencing technologies (RNA-seq). This introduction describes normalization and analysis of data generated from microarray or RNA-seq experiments.

  20. MicroRNA expression profiling using microarrays.

    PubMed

    Love, Cassandra; Dave, Sandeep

    2013-01-01

    MicroRNAs are small noncoding RNAs which are able to regulate gene expression at both the transcriptional and translational levels. There is a growing recognition of the role of microRNAs in nearly every tissue type and cellular process. Thus there is an increasing need for accurate quantitation of microRNA expression in a variety of tissues. Microarrays provide a robust method for the examination of microRNA expression. In this chapter, we describe detailed methods for the use of microarrays to measure microRNA expression and discuss methods for the analysis of microRNA expression data.

  1. Metric learning for DNA microarray data analysis

    NASA Astrophysics Data System (ADS)

    Takeuchi, Ichiro; Nakagawa, Masao; Seto, Masao

    2009-12-01

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

  2. [DNA microarrays in parasitology and medical sciences].

    PubMed

    Jaros, Sławomir

    2006-01-01

    The article presents the current knowledge on the microarray technique and its applications in medical sciences and parasitology. The first part of the article is focused on the technical aspects (microarray preparation, different microarray platforms, probes preparation, hybridization and signal detection). The article also describes possible ways of proceeding during laboratory work on organism of which the genome sequence is not known or has been only partially sequenced. The second part of the review describes how microarray technique have been, or possibly will be, used for better understanding parasite life cycles and development, host-parasite relationship, comparative genomics of virulent organisms, develpoment vaccines against the most virulent parasites and host responses to infection.

  3. DNA microarray technology for the microbiologist: an overview.

    PubMed

    Ehrenreich, Armin

    2006-11-01

    DNA microarrays have found widespread use as a flexible tool to investigate bacterial metabolism. Their main advantage is the comprehensive data they produce on the transcriptional response of the whole genome to an environmental or genetic stimulus. This allows the microbiologist to monitor metabolism and to define stimulons and regulons. Other fields of application are the identification of microorganisms or the comparison of genomes. The importance of this technology increases with the number of sequenced genomes and the falling prices for equipment and oligonucleotides. Knowledge of DNA microarrays is of rising relevance for many areas in microbiological research. Much literature has been published on various specific aspects of this technique that can be daunting to the casual user and beginner. This article offers a comprehensive outline of microarray technology for transcription analysis in microbiology. It shortly discusses the types of DNA microarrays available, the printing of custom arrays, common labeling strategies for targets, hybridization, scanning, normalization, and clustering of expression data.

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

    SciTech Connect

    Choi, Yoo Seong; Pack, Seung Pil; Yoo, Young Je . E-mail: yjyoo@snu.ac.kr

    2005-04-22

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

  5. Robust and efficient synthetic method for forming DNA microarrays.

    PubMed

    Dolan, P L; Wu, Y; Ista, L K; Metzenberg, R L; Nelson, M A; Lopez, G P

    2001-11-01

    The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

  6. Statistical approaches for the analysis of DNA methylation microarray data.

    PubMed

    Siegmund, Kimberly D

    2011-06-01

    Following the rapid development and adoption in DNA methylation microarray assays, we are now experiencing a growth in the number of statistical tools to analyze the resulting large-scale data sets. As is the case for other microarray applications, biases caused by technical issues are of concern. Some of these issues are old (e.g., two-color dye bias and probe- and array-specific effects), while others are new (e.g., fragment length bias and bisulfite conversion efficiency). Here, I highlight characteristics of DNA methylation that suggest standard statistical tools developed for other data types may not be directly suitable. I then describe the microarray technologies most commonly in use, along with the methods used for preprocessing and obtaining a summary measure. I finish with a section describing downstream analyses of the data, focusing on methods that model percentage DNA methylation as the outcome, and methods for integrating DNA methylation with gene expression or genotype data.

  7. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    PubMed

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  8. Automated DNA profile analysis.

    PubMed

    Graham, Eleanor A M

    2005-12-01

    DNA profile analysis is not a simple process. Stringent demands are placed on the accuracy and consistency of forensic evidence so that complex, robust, and reproducible guidelines are necessary to assist the analyst and ensure mistakes are eliminated before a final profile is reported. The guidelines used for forensic DNA profile interpretation are formulated by investigation and statistical evaluation of all aspects of the analytical procedure. All the resulting rules, formulas, and thresholds are perfectly suited to programming of "expert systems"-software programs that imitate the human expert in decision-based processes to formulate a conclusion. Expert systems in forensic DNA analysis will contribute greatly to this field by increasing analytical throughput. The net result of this will be an increase in the human resources available for the research and development of improved methodologies, to ensure that forensic DNA profiling continues to advance at its current impressive rate.

  9. In Situ-Synthesized Novel Microarray Optimized for Mouse Stem Cell and Early Developmental Expression Profiling

    PubMed Central

    Carter, Mark G.; Hamatani, Toshio; Sharov, Alexei A.; Carmack, Condie E.; Qian, Yong; Aiba, Kazuhiro; Ko, Naomi T.; Dudekula, Dawood B.; Brzoska, Pius M.; Hwang, S. Stuart; Ko, Minoru S.H.

    2003-01-01

    Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research. [Supplemental material is available online at www.genome.org and at the NIA Mouse cDNA Project Web site, http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html.] PMID:12727912

  10. Molecular diagnosis and prognosis with DNA microarrays.

    PubMed

    Wiltgen, Marco; Tilz, Gernot P

    2011-05-01

    Microarray analysis makes it possible to determine thousands of gene expression values simultaneously. Changes in gene expression, as a response to diseases, can be detected allowing a better understanding and differentiation of diseases at a molecular level. By comparing different kinds of tissue, for example healthy tissue and cancer tissue, the microarray analysis indicates induced gene activity, repressed gene activity or when there is no change in the gene activity level. Fundamental patterns in gene expression are extracted by several clustering and machine learning algorithms. Certain kinds of cancer can be divided into subtypes, with different clinical outcomes, by their specific gene expression patterns. This enables a better diagnosis and tailoring of individual patient treatments.

  11. DNA Microarray Detection of 18 Important Human Blood Protozoan Species.

    PubMed

    Chen, Mu-Xin; Ai, Lin; Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

    2016-12-01

    Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species.

  12. DNA Microarray Detection of 18 Important Human Blood Protozoan Species

    PubMed Central

    Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

    2016-01-01

    Background Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. Methodology/Principal Findings The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Conclusions/Significance Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species. PMID:27911895

  13. Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach

    PubMed Central

    Pan, Yi-Shin; Lee, Yun-Shien; Lee, Yung-Lin; Lee, Wei-Chen; Hsieh, Sen-Yung

    2006-01-01

    Background The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. Results We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma

  14. Performance comparison of SLFN training algorithms for DNA microarray classification.

    PubMed

    Huynh, Hieu Trung; Kim, Jung-Ja; Won, Yonggwan

    2011-01-01

    The classification of biological samples measured by DNA microarrays has been a major topic of interest in the last decade, and several approaches to this topic have been investigated. However, till now, classifying the high-dimensional data of microarrays still presents a challenge to researchers. In this chapter, we focus on evaluating the performance of the training algorithms of the single hidden layer feedforward neural networks (SLFNs) to classify DNA microarrays. The training algorithms consist of backpropagation (BP), extreme learning machine (ELM) and regularized least squares ELM (RLS-ELM), and an effective algorithm called neural-SVD has recently been proposed. We also compare the performance of the neural network approaches with popular classifiers such as support vector machine (SVM), principle component analysis (PCA) and fisher discriminant analysis (FDA).

  15. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization1

    PubMed Central

    Beheshti, Ben; Braude, Ilan; Marrano, Paula; Thorner, Paul; Zielenska, Maria; Squire, Jeremy A

    2003-01-01

    Abstract Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma. PMID:12659670

  16. A comparative analysis of DNA barcode microarray feature size

    PubMed Central

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

    2009-01-01

    Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 μm features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 μm feature size is of comparable quality to the 30 μm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density. PMID:19825181

  17. Application of click chemistry to the production of DNA microarrays.

    PubMed

    Uszczyńska, Barbara; Ratajczak, Tomasz; Frydrych, Emilia; Maciejewski, Hieronim; Figlerowicz, Marek; Markiewicz, Wojciech T; Chmielewski, Marcin K

    2012-03-21

    The copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was applied as the novel method of DNA immobilization on a modified solid support. The CuAAC click reaction enables the covalent binding of DNA modified with pentynyl groups at its 5'-end to azide-loaded slides. Click microarrays were produced using this approach and successfully employed in biological/model experiments.

  18. PNA microarrays for hybridisation of unlabelled DNA samples

    PubMed Central

    Brandt, Ole; Feldner, Julia; Stephan, Achim; Schröder, Markus; Schnölzer, Martina; Arlinghaus, Heinrich F.; Hoheisel, Jörg D.; Jacob, Anette

    2003-01-01

    Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces. PMID:14500847

  19. Gene expression analysis of perennial ryegrass (Lolium perenne) using cDNA microarrays

    NASA Astrophysics Data System (ADS)

    Ong, Eng-Kok; Sawbridge, Tim; Webster, Tracie; Emmerling, Michael; Nguyen, Nga; Nunan, Katrina; O'Neill, Matthew; O'Toole, Fiona; Rhodes, Carolyn; Simmonds, Jason; Tian, Pei; Wearne, Katherine; Winkworth, Amanda; Spangenberg, German

    2003-07-01

    Perennial ryegrass (Lolium perenne) is a major forage grass of temperate pastures. A genomics program has been undertaken generating over 52,000 expressed sequence tags (ESTs). Cluster analysis of the ESTs identified approximately 14,600 ryegrass unigenes. In this report, we described the application of ryegrass unigene cDNAs to produce ryegrass 15K microarray. Fifteen microarray hybridisations were performed with labeled total RNA isolated from a variety of plant organs and developmental stages. In a proof of concept, gene expression profiling of ryegrass ESTs using the 15K unigene microarrays has been established using several known genes and two cluster analysis approaches (parallel coordinate planes plot and hierarchical clustering). The expression profile of the known genes (e.g. rubisco and invertase) corresponds well with published data. The microarray expression profile of a ryegrass putative root specific kinase gene was also verified with Northern blotting. This combination of DNA microarray hybridisations and cluster analysis can be applied as a tool for the identification of novel sequences of unknown function.

  20. Unravelling Microbial Communities with DNA-Microarrays: Challengesand Future Directions.

    SciTech Connect

    Wagner, Michael; Smidt, Hauke; Loy, Alexander; Zhou, Jizhong

    2007-03-08

    High-throughput technologies are urgently needed formonitoring the formidable biodiversity and functional capabilities ofmicroorganisms in the environment. Ten years ago, DNA microarrays,miniaturized platforms for highly parallel hybridization reactions, foundtheir way into environmental microbiology and raised great expectationsamong researchers in the field. In this article, we briefly summarize thestate-of-the-art of microarray approaches in microbial ecology researchand discuss in more detail crucial problems and promising solutions.Finally, we outline scenarios for an innovative combination ofmicroarrays with other molecular tools for structure-function analysis ofcomplex microbial communities.

  1. A Customized DNA Microarray for Microbial Source Tracking ...

    EPA Pesticide Factsheets

    It is estimated that more than 160, 000 miles of rivers and streams in the United States are impaired due to the presence of waterborne pathogens. These pathogens typically originate from human and other animal fecal pollution sources; therefore, a rapid microbial source tracking (MST) method is needed to facilitate water quality assessment and impaired water remediation. We report a novel qualitative DNA microarray technology consisting of 453 probes for the detection of general fecal and host-associated bacteria, viruses, antibiotic resistance, and other environmentally relevant genetic indicators. A novel data normalization and reduction approach is also presented to help alleviate false positives often associated with high-density microarray applications. To evaluate the performance of the approach, DNA and cDNA was isolated from swine, cattle, duck, goose and gull fecal reference samples, as well as soiled poultry liter and raw municipal sewage. Based on nonmetric multidimensional scaling analysis of results, findings suggest that the novel microarray approach may be useful for pathogen detection and identification of fecal contamination in recreational waters. The ability to simultaneously detect a large collection of environmentally important genetic indicators in a single test has the potential to provide water quality managers with a wide range of information in a short period of time. Future research is warranted to measure microarray performance i

  2. A Customized DNA Microarray for Microbial Source Tracking ...

    EPA Pesticide Factsheets

    It is estimated that more than 160, 000 miles of rivers and streams in the United States are impaired due to the presence of waterborne pathogens. These pathogens typically originate from human and other animal fecal pollution sources; therefore, a rapid microbial source tracking (MST) method is needed to facilitate water quality assessment and impaired water remediation. We report a novel qualitative DNA microarray technology consisting of 453 probes for the detection of general fecal and host-associated bacteria, viruses, antibiotic resistance, and other environmentally relevant genetic indicators. A novel data normalization and reduction approach is also presented to help alleviate false positives often associated with high-density microarray applications. To evaluate the performance of the approach, DNA and cDNA was isolated from swine, cattle, duck, goose and gull fecal reference samples, as well as soiled poultry liter and raw municipal sewage. Based on nonmetric multidimensional scaling analysis of results, findings suggest that the novel microarray approach may be useful for pathogen detection and identification of fecal contamination in recreational waters. The ability to simultaneously detect a large collection of environmentally important genetic indicators in a single test has the potential to provide water quality managers with a wide range of information in a short period of time. Future research is warranted to measure microarray performance i

  3. Fecal source tracking in water using a mitochondrial DNA microarray.

    PubMed

    Vuong, Nguyet-Minh; Villemur, Richard; Payment, Pierre; Brousseau, Roland; Topp, Edward; Masson, Luke

    2013-01-01

    A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

  4. Deoxyoligonucleotide microarrays for gene expression profiling in murine tooth germs.

    PubMed

    Osmundsen, Harald; Jevnaker, Anne-Marthe; Landin, Maria A

    2012-01-01

    The use of deoxyoligonucleotide microarrays facilitates rapid expression profiling of gene expression using samples of about 1 μg of total RNA. Here are described practical aspects of the procedures involved, including essential reagents. Analysis of results is discussed from a practical, experimental, point of view together with software required to carry out the required statistical analysis to isolate populations of differentially expressed genes.

  5. Do DNA Microarrays Tell the Story of Gene Expression?

    PubMed Central

    Rosenfeld, Simon

    2010-01-01

    Poor reproducibility of microarray measurements is a major obstacle to their application as an instrument for clinical diagnostics. In this paper, several aspects of poor reproducibility are analyzed. All of them belong to the category of interpretive weaknesses of DNA microarray technology. First, the attention is drawn to the fact that absence of the information regarding post-transcriptional mRNA stability makes it impossible to evaluate the level of gene activity from the relative mRNA abundances, the quantities available from microarray measurements. Second, irreducible intracellular variability with persistent patterns of stochasticity and burstiness put natural limits to reproducibility. Third, strong interactions within intracellular biomolecular networks make it highly problematic to build a bridge between transcription rates of individual genes and structural fidelity of their genetic codes. For these reasons, the microarray measurements of relative mRNA abundances are more appropriate in laboratory settings as a tool for scientific research, hypotheses generating and producing the leads for subsequent validation through more sophisticated technologies. As to clinical settings, where firm conclusive diagnoses, not the leads for further experimentation, are required, microarrays still have a long way to go until they become a reliable instrument in patient-related decision making. PMID:20628535

  6. Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

    PubMed

    Yang, Dan-Hui; Barari, Mehrnoosh; Arif, Basil M; Krell, Peter J

    2007-08-01

    A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

  7. Increasing hybridization rate and sensitivity of DNA microarrays using isotachophoresis.

    PubMed

    Han, Crystal M; Katilius, Evaldas; Santiago, Juan G

    2014-08-21

    We present an on-chip electrokinetic method to increase the reaction kinetics and sensitivity of DNA microarray hybridization. We use isotachophoresis (ITP) to preconcentrate target molecules in solution and transport them over the immobilized probe sites of a microarray, greatly increasing the binding reaction rate. We show theoretically and experimentally that ITP-enhanced microarrays can be hybridized much faster and with higher sensitivity than conventional methods. We demonstrate our assay using a microfluidic system consisting of a PDMS microchannel superstructure bonded onto a glass slide on which 60 spots of 20-27 nt ssDNA oligonucleotide probes are immobilized. Our 30 min assay results in an 8.2 fold higher signal than the conventional overnight hybridization at 100 fM target concentration. We show rapid and quantitative detection over 4 orders of magnitude dynamic range of target concentration with no increase in the nonspecific signal. Our technique can be further multiplexed for higher density microarrays and extended for other reactions of target-surface immobilized ligands.

  8. Viral Discovery and Sequence Recovery Using DNA Microarrays

    PubMed Central

    Wang, David; Urisman, Anatoly; Liu, Yu-Tsueng; Springer, Michael; Ksiazek, Thomas G; Erdman, Dean D; Mardis, Elaine R; Hickenbotham, Matthew; Magrini, Vincent; Eldred, James; Latreille, J. Phillipe; Wilson, Richard K; Ganem, Don

    2003-01-01

    Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease. PMID:14624234

  9. Impact of surface chemistry and blocking strategies on DNA microarrays.

    PubMed

    Taylor, Scott; Smith, Stephanie; Windle, Brad; Guiseppi-Elie, Anthony

    2003-08-15

    The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.

  10. Scanometric analysis of DNA microarrays using DNA intercalator-conjugated gold nanoparticles.

    PubMed

    Cho, Hyunmin; Jung, Juyeon; Chung, Bong Hyun

    2012-08-07

    We introduce a scanometric detection method for the analysis of DNA microarrays using DNA intercalator-conjugated gold nanoparticles that can be analyzed with the naked eye or with an optical scanner after the enhancement of the AuNPs. Moreover, we successfully detected a hemagglutinin-subtyping DNA array using this method.

  11. Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

    PubMed Central

    Guimond, Chantal; Trudel, Nathalie; Brochu, Christian; Marquis, Nathalie; Fadili, Amal El; Peytavi, Régis; Briand, Guylaine; Richard, Dave; Messier, Nadine; Papadopoulou, Barbara; Corbeil, Jacques; Bergeron, Michel G.; Légaré, Danielle; Ouellette, Marc

    2003-01-01

    In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance. PMID:14530437

  12. Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays.

    PubMed

    Guimond, Chantal; Trudel, Nathalie; Brochu, Christian; Marquis, Nathalie; El Fadili, Amal; Peytavi, Régis; Briand, Guylaine; Richard, Dave; Messier, Nadine; Papadopoulou, Barbara; Corbeil, Jacques; Bergeron, Michel G; Légaré, Danielle; Ouellette, Marc

    2003-10-15

    In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.

  13. Glycan profiling of endometrial cancers using lectin microarray.

    PubMed

    Nishijima, Yoshihiro; Toyoda, Masashi; Yamazaki-Inoue, Mayu; Sugiyama, Taro; Miyazawa, Masaki; Muramatsu, Toshinari; Nakamura, Kyoko; Narimatsu, Hisashi; Umezawa, Akihiro; Mikami, Mikio

    2012-10-01

    Cell surface glycans change during the process of malignant transformation. To characterize and distinguish endometrial cancer and endometrium, we performed glycan profiling using an emerging modern technology, lectin microarray analysis. The three cell lines, two from endometrial cancers [well-differentiated type (G1) and poorly differentiated type (G3)] and one from normal endometrium, were successfully categorized into three independent groups by 45 lectins. Furthermore, in cancer cells, a clear difference between G1 and G3 type was observed for the glycans recognized with six lectins, Ulex europaeus agglutinin I (UEA-I), Sambucus sieboldiana agglutinin (SSA), Sambucus nigra agglutinin (SNA), Trichosanthes japonica agglutinin I (TJA-I), Amaranthus caudatus agglutinin (ACA), and Bauhinia purpurea lectin (BPL). The lectin microarray analysis using G3 type tissues demonstrated that stage I and stage III or IV were distinguished depending on signal pattern of three lectins, Dolichos biflorus agglutinin (DBA), BPL, and ACA. In addition, the analysis of the glycans on the ovarian cancer cells showed that only anticancer drug-sensitive cell lines had almost no activities to specific three lectins. Glycan profiling by the lectin microarray may be used to assess the characteristics of tumors and potentially to predict the success of chemotherapy treatment.

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

    SciTech Connect

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

    2002-01-01

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

  15. Detection and Identification of Mycobacterium Species Isolates by DNA Microarray

    PubMed Central

    Fukushima, Masao; Kakinuma, Kenichi; Hayashi, Hiroshi; Nagai, Hiroko; Ito, Kunihiko; Kawaguchi, Ryuji

    2003-01-01

    Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis. PMID:12791887

  16. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.

    PubMed

    Warsen, Adelaide E; Krug, Melissa J; LaFrentz, Stacey; Stanek, Danielle R; Loge, Frank J; Call, Douglas R

    2004-07-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

  17. Microarray analysis of gene expression profiles in ripening pineapple fruits.

    PubMed

    Koia, Jonni H; Moyle, Richard L; Botella, Jose R

    2012-12-18

    Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit

  18. Microarray analysis of gene expression profiles in ripening pineapple fruits

    PubMed Central

    2012-01-01

    Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the

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

    PubMed

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

    2000-11-15

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

  20. Profiling genome-wide DNA methylation.

    PubMed

    Yong, Wai-Shin; Hsu, Fei-Man; Chen, Pao-Yang

    2016-01-01

    DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

  1. DNA microarrays: translation of the genome from laboratory to clinic.

    PubMed

    Geschwind, Daniel H

    2003-05-01

    As the complete sequences of human and other mammalian genomes become available we are faced with the challenge of understanding how variation in sequence and gene expression contributes to neurological and psychiatric disorders. DNA microarrays, or DNA chips, provide the means to measure simultaneously where and when thousands of genes are expressed. Microarrays are changing the way that researchers approach work at the bench and have already yielded new insights into brain tumours, multiple sclerosis, acute neurological insults such as stroke and seizures, and schizophrenia. The study of disease-related changes in gene expression is the first step in the long process in translation of genome research to the clinic. Eventually, the changes observed in microarray studies will need to be independently confirmed and we wil need to understand how gene expression changes translate into functional effects at the cellular level in the nervous system. Progress in these studies will translate into array-based disease classification schemes and help optimise therapy for individual patients based on gene expression patterns or their genetic background.

  2. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  3. An antibody profile of systemic lupus erythematosus detected by antigen microarray

    PubMed Central

    Fattal, Ittai; Shental, Noam; Mevorach, Dror; Anaya, Juan-Manuel; Livneh, Avi; Langevitz, Pnina; Zandman-Goddard, Gisele; Pauzner, Rachel; Lerner, Miriam; Blank, Miri; Hincapie, Maria-Eugenia; Gafter, Uzi; Naparstek, Yaakov; Shoenfeld, Yehuda; Domany, Eytan; Cohen, Irun R

    2010-01-01

    Patients with systemic lupus erythematosus (SLE) produce antibodies to many different self-antigens. Here, we investigated antibodies in SLE sera using an antigen microarray containing many hundreds of antigens, mostly self-antigens. The aim was to detect sets of antibody reactivities characteristic of SLE patients in each of various clinical states – SLE patients with acute lupus nephritis, SLE patients in renal remission, and SLE patients who had never had renal involvement. The analysis produced two novel findings: (i) an SLE antibody profile persists independently of disease activity and despite long-term clinical remission, and (ii) this SLE antibody profile includes increases in four specific immunoglobulin G (IgG) reactivities to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), Epstein–Barr virus (EBV) and hyaluronic acid; the profile also includes decreases in specific IgM reactivities to myeloperoxidase (MPO), CD99, collagen III, insulin-like growth factor binding protein 1 (IGFBP1) and cardiolipin. The reactivities together showed high sensitivity (> 93%) and high specificity for SLE (> 88%). A healthy control subject who had the SLE antibody profile was later found to develop clinical SLE. The present study did not detect antibody reactivities that differentiated among the various subgroups of SLE subjects with statistical significance. Thus, SLE is characterized by an enduring antibody profile irrespective of clinical state. The association of SLE with decreased IgM natural autoantibodies suggests that these autoantibodies might enhance resistance to SLE. PMID:20201986

  4. [Legal implication of DNA profiling].

    PubMed

    Doutremepuich, Christian

    2012-06-01

    In recent years, DNA profiling has been used regularly by the justice system, and has seen a number of improvements, with the need for fewer cells, more efficient DNA extraction and purification, and more rapid genotyping. These methods can now identify an individual more rapidly, from a corpse, blood stain, sperm or epithelial cells, by comparison with familial profiles. In France, DNA profiling can only be ordered by a judge.

  5. Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression

    PubMed Central

    Rhodes, Daniel R.; Yu, Jianjun; Shanker, K.; Deshpande, Nandan; Varambally, Radhika; Ghosh, Debashis; Barrette, Terrence; Pandey, Akhilesh; Chinnaiyan, Arul M.

    2004-01-01

    Many studies have used DNA microarrays to identify the gene expression signatures of human cancer, yet the critical features of these often unmanageably large signatures remain elusive. To address this, we developed a statistical method, comparative metaprofiling, which identifies and assesses the intersection of multiple gene expression signatures from a diverse collection of microarray data sets. We collected and analyzed 40 published cancer microarray data sets, comprising 38 million gene expression measurements from >3,700 cancer samples. From this, we characterized a common transcriptional profile that is universally activated in most cancer types relative to the normal tissues from which they arose, likely reflecting essential transcriptional features of neoplastic transformation. In addition, we characterized a transcriptional profile that is commonly activated in various types of undifferentiated cancer, suggesting common molecular mechanisms by which cancer cells progress and avoid differentiation. Finally, we validated these transcriptional profiles on independent data sets. PMID:15184677

  6. A glance at DNA microarray technology and applications.

    PubMed

    Saei, Amir Ata; Omidi, Yadollah

    2011-01-01

    Because of huge impacts of "OMICS" technologies in life sciences, many researchers aim to implement such high throughput approach to address cellular and/or molecular functions in response to any influential intervention in genomics, proteomics, or metabolomics levels. However, in many cases, use of such technologies often encounters some cybernetic difficulties in terms of knowledge extraction from a bunch of data using related softwares. In fact, there is little guidance upon data mining for novices. The main goal of this article is to provide a brief review on different steps of microarray data handling and mining for novices and at last to introduce different PC and/or web-based softwares that can be used in preprocessing and/or data mining of microarray data. To pursue such aim, recently published papers and microarray softwares were reviewed. It was found that defining the true place of the genes in cell networks is the main phase in our understanding of programming and functioning of living cells. This can be obtained with global/selected gene expression profiling. Studying the regulation patterns of genes in groups, using clustering and classification methods helps us understand different pathways in the cell, their functions, regulations and the way one component in the system affects the other one. These networks can act as starting points for data mining and hypothesis generation, helping us reverse engineer.

  7. A Glance at DNA Microarray Technology and Applications

    PubMed Central

    Saei, Amir Ata; Omidi, Yadollah

    2011-01-01

    Introduction Because of huge impacts of “OMICS” technologies in life sciences, many researchers aim to implement such high throughput approach to address cellular and/or molecular functions in response to any influential intervention in genomics, proteomics, or metabolomics levels. However, in many cases, use of such technologies often encounters some cybernetic difficulties in terms of knowledge extraction from a bunch of data using related softwares. In fact, there is little guidance upon data mining for novices. The main goal of this article is to provide a brief review on different steps of microarray data handling and mining for novices and at last to introduce different PC and/or web-based softwares that can be used in preprocessing and/or data mining of microarray data. Methods To pursue such aim, recently published papers and microarray softwares were reviewed. Results It was found that defining the true place of the genes in cell networks is the main phase in our understanding of programming and functioning of living cells. This can be obtained with global/selected gene expression profiling. Conclusion Studying the regulation patterns of genes in groups, using clustering and classification methods helps us understand different pathways in the cell, their functions, regulations and the way one component in the system affects the other one. These networks can act as starting points for data mining and hypothesis generation, helping us reverse engineer. PMID:23678411

  8. Monitoring of dnaK gene expression in Porphyromonas gingivalis by oxygen stress using DNA microarray.

    PubMed

    Araki, Makoto; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Abiko, Yoshimitsu

    2004-06-01

    Porphyromonas gingivalis, a Gram-negative anaerobe associated with adult periodontitis, expresses numerous potential virulence factors. dnaK, a member of the heat shock protein family, functions as a molecular chaperone and plays a role in microbial pathogenicity. However, little is known regarding its gene expression caused by oxygen stress in P. gingivalis. In the present study, a custom-made DNA microarray was designed and used to monitor dnaK gene expression in P. gingivalis caused by oxygen stress. The results demonstrated that dnaK mRNA was up-regulated in a short time, and the DNA microarray results were confirmed by real-time polymerase chain reaction analysis. These findings suggest that oxygen stress stimulates gene expression of dnaK and may have a relationship to the aerotolerance activity of this organism as well as its expression of pathogenesis.

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

    PubMed

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

    2007-04-01

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

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

    ERIC Educational Resources Information Center

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

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH…

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

    ERIC Educational Resources Information Center

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

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH…

  12. [Software development in data analysis and mining for cDNA microarray].

    PubMed

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

  13. Microarray expression profiling in adhesion and normal peritoneal tissues.

    PubMed

    Ambler, Dana R; Golden, Alicia M; Gell, Jennifer S; Saed, Ghassan M; Carey, David J; Diamond, Michael P

    2012-05-01

    To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. Comparative study. University hospital. Five premenopausal women. Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. Gene expression in postoperative adhesion and normal peritoneal tissues. A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. A dynamic bead-based microarray for parallel DNA detection

    NASA Astrophysics Data System (ADS)

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

    2011-05-01

    A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

  15. Feature extraction and signal processing for nylon DNA microarrays

    PubMed Central

    Lopez, F; Rougemont, J; Loriod, B; Bourgeois, A; Loï, L; Bertucci, F; Hingamp, P; Houlgatte, R; Granjeaud, S

    2004-01-01

    Background High-density DNA microarrays require automatic feature extraction methodologies and softwares. These can be a potential source of non-reproducibility of gene expression measurements. Variation in feature location or in signal integration methodology may be a significant contribution to the observed variance in gene expression levels. Results We explore sources of variability in feature extraction from DNA microarrays on Nylon membrane with radioactive detection. We introduce a mathematical model of the signal emission and derive methods for correcting biases such as overshining, saturation or variation in probe amount. We also provide a quality metric which can be used qualitatively to flag weak or untrusted signals or quantitatively to modulate the weight of each experiment or gene in higher level analyses (clustering or discriminant analysis). Conclusions Our novel feature extraction methodology, based on a mathematical model of the radioactive emission, reduces variability due to saturation, neighbourhood effects and variable probe amount. Furthermore, we provide a fully automatic feature extraction software, BZScan, which implements the algorithms described in this paper. PMID:15222896

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

    NASA Astrophysics Data System (ADS)

    Tchagang, Alain B.; Tewfik, Ahmed H.

    2006-12-01

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

  17. Bacterial DNA microarrays for clinical microbiology: the early logarithmic phase.

    PubMed

    Cassone, Marco; Giordano, Antonio; Pozzi, Gianni

    2007-01-01

    In this era of coexistence of high-throughput sequencing technologies and serious difficulties in the management of both common and novel infectious syndromes, new techniques which improve the study of micro-organisms is timely. In bacteriology, the most important subjects are bacterial pathogenicity, discovery of the genomic complexity of bacteria, and the epidemiology of antimicrobial resistance traits. From the clinical point of view, genetic testing is flanking phenotypic testing for the assessment of new, difficult to test antibiotic resistance traits, and for correlations with the microbial behaviour in vivo. The demand for faster, comprehensive and highly parallel microbial diagnostics is also cogent even at the basic laboratory level, where the ultimate objective is saving lives. In this setting, DNA microarrays offer a pivotal contribution by allowing performance of hybridization experiments in highly parallel formats, with an increasing reliability. Not only they are useful in deciphering host and microbial pathophysiology, they can also make the difference in the management of prognostic and therapeutic aspects of many diseases. Here, we provide an overview of the current use and the potential of DNA microarrays in clinical bacteriology, and several applications and technical solutions are discussed.

  18. [DNA microarrays and their application in detecting and identifying intestinal pathogens].

    PubMed

    Jin, Da-Zhi; Wen, Si-Yuan; Wang, Sheng-Qi

    2006-06-01

    DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

  19. [DNA microarrays (DNA chips) used in molecular medical research].

    PubMed

    Ørntoft, Torben F

    2003-02-17

    Microchip technology and mapping of the human genome has led to the invention of DNA-micro-array technology. In that technology thousands of genes are analysed in parallel on a small glass surface. The very large data material that is generated requires bioinformatic analysis in order to be transformed to understandable biological results. There are different forms of DNA-micro-arrays. Some are used for measuring gene expression, i.e. which genes are turned on and which genes are turned off. Others are used for sequencing, and finally some are used for measuring single nucleotide polymorphisms (SNPs). Some application possibilities of the arrays within the area of cell biology and classification of diseases are presented. Especially within the area of classification of cancer diseases the technology is promising, and a scientific breakthrough could occur in the area of classification of very aggressive cancers.

  20. DFP: a Bioconductor package for fuzzy profile identification and gene reduction of microarray data

    PubMed Central

    Glez-Peña, Daniel; Álvarez, Rodrigo; Díaz, Fernando; Fdez-Riverola, Florentino

    2009-01-01

    Background Expression profiling assays done by using DNA microarray technology generate enormous data sets that are not amenable to simple analysis. The greatest challenge in maximizing the use of this huge amount of data is to develop algorithms to interpret and interconnect results from different genes under different conditions. In this context, fuzzy logic can provide a systematic and unbiased way to both (i) find biologically significant insights relating to meaningful genes, thereby removing the need for expert knowledge in preliminary steps of microarray data analyses and (ii) reduce the cost and complexity of later applied machine learning techniques being able to achieve interpretable models. Results DFP is a new Bioconductor R package that implements a method for discretizing and selecting differentially expressed genes based on the application of fuzzy logic. DFP takes advantage of fuzzy membership functions to assign linguistic labels to gene expression levels. The technique builds a reduced set of relevant genes (FP, Fuzzy Pattern) able to summarize and represent each underlying class (pathology). A last step constructs a biased set of genes (DFP, Discriminant Fuzzy Pattern) by intersecting existing fuzzy patterns in order to detect discriminative elements. In addition, the software provides new functions and visualisation tools that summarize achieved results and aid in the interpretation of differentially expressed genes from multiple microarray experiments. Conclusion DFP integrates with other packages of the Bioconductor project, uses common data structures and is accompanied by ample documentation. It has the advantage that its parameters are highly configurable, facilitating the discovery of biologically relevant connections between sets of genes belonging to different pathologies. This information makes it possible to automatically filter irrelevant genes thereby reducing the large volume of data supplied by microarray experiments. Based on

  1. DFP: a Bioconductor package for fuzzy profile identification and gene reduction of microarray data.

    PubMed

    Glez-Peña, Daniel; Alvarez, Rodrigo; Díaz, Fernando; Fdez-Riverola, Florentino

    2009-01-29

    Expression profiling assays done by using DNA microarray technology generate enormous data sets that are not amenable to simple analysis. The greatest challenge in maximizing the use of this huge amount of data is to develop algorithms to interpret and interconnect results from different genes under different conditions. In this context, fuzzy logic can provide a systematic and unbiased way to both (i) find biologically significant insights relating to meaningful genes, thereby removing the need for expert knowledge in preliminary steps of microarray data analyses and (ii) reduce the cost and complexity of later applied machine learning techniques being able to achieve interpretable models. DFP is a new Bioconductor R package that implements a method for discretizing and selecting differentially expressed genes based on the application of fuzzy logic. DFP takes advantage of fuzzy membership functions to assign linguistic labels to gene expression levels. The technique builds a reduced set of relevant genes (FP, Fuzzy Pattern) able to summarize and represent each underlying class (pathology). A last step constructs a biased set of genes (DFP, Discriminant Fuzzy Pattern) by intersecting existing fuzzy patterns in order to detect discriminative elements. In addition, the software provides new functions and visualisation tools that summarize achieved results and aid in the interpretation of differentially expressed genes from multiple microarray experiments. DFP integrates with other packages of the Bioconductor project, uses common data structures and is accompanied by ample documentation. It has the advantage that its parameters are highly configurable, facilitating the discovery of biologically relevant connections between sets of genes belonging to different pathologies. This information makes it possible to automatically filter irrelevant genes thereby reducing the large volume of data supplied by microarray experiments. Based on these contributions GENECBR, a

  2. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

    PubMed Central

    Bulyk, Martha L.; Huang, Xiaohua; Choo, Yen; Church, George M.

    2001-01-01

    A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites. PMID:11404456

  3. Optimized LOWESS normalization parameter selection for DNA microarray data

    PubMed Central

    Berger, John A; Hautaniemi, Sampsa; Järvinen, Anna-Kaarina; Edgren, Henrik; Mitra, Sanjit K; Astola, Jaakko

    2004-01-01

    Background Microarray data normalization is an important step for obtaining data that are reliable and usable for subsequent analysis. One of the most commonly utilized normalization techniques is the locally weighted scatterplot smoothing (LOWESS) algorithm. However, a much overlooked concern with the LOWESS normalization strategy deals with choosing the appropriate parameters. Parameters are usually chosen arbitrarily, which may reduce the efficiency of the normalization and result in non-optimally normalized data. Thus, there is a need to explore LOWESS parameter selection in greater detail. Results and discussion In this work, we discuss how to choose parameters for the LOWESS method. Moreover, we present an optimization approach for obtaining the fraction of data points utilized in the local regression and analyze results for local print-tip normalization. The optimization procedure determines the bandwidth parameter for the local regression by minimizing a cost function that represents the mean-squared difference between the LOWESS estimates and the normalization reference level. We demonstrate the utility of the systematic parameter selection using two publicly available data sets. The first data set consists of three self versus self hybridizations, which allow for a quantitative study of the optimization method. The second data set contains a collection of DNA microarray data from a breast cancer study utilizing four breast cancer cell lines. Our results show that different parameter choices for the bandwidth window yield dramatically different calibration results in both studies. Conclusions Results derived from the self versus self experiment indicate that the proposed optimization approach is a plausible solution for estimating the LOWESS parameters, while results from the breast cancer experiment show that the optimization procedure is readily applicable to real-life microarray data normalization. In summary, the systematic approach to obtain critical

  4. Design issues in toxicogenomics using DNA microarray experiment

    SciTech Connect

    Lee, Kyoung-Mu; Kim, Ju-Han; Kang, Daehee . E-mail: dhkang@snu.ac.kr

    2005-09-01

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

  5. Automated and Multiplexed Soft Lithography for the Production of Low-Density DNA Microarrays

    PubMed Central

    Fredonnet, Julie; Foncy, Julie; Cau, Jean-Christophe; Séverac, Childérick; François, Jean Marie; Trévisiol, Emmanuelle

    2016-01-01

    Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40®. This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStampTM bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStampTM were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays. PMID:27681742

  6. Structural analysis of hepatitis C RNA genome using DNA microarrays

    PubMed Central

    Martell, María; Briones, Carlos; de Vicente, Aránzazu; Piron, María; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gómez, Jordi

    2004-01-01

    Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5′ non-coding region (5′NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions. PMID:15247323

  7. Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Biyani, Manish; Moriyasu, Junpei; Tanaka, Yoko; Sato, Shusuke; Ueno, Shingo; Ichiki, Takanori

    2013-08-01

    A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25-100 µm features.

  8. Fabrication of high quality cDNA microarray using a small amount of cDNA.

    PubMed

    Park, Chan Hee; Jeong, Ha Jin; Jung, Jae Jun; Lee, Gui Yeon; Kim, Sang-Chul; Kim, Tae Soo; Yang, Sang Hwa; Chung, Hyun Cheol; Rha, Sun Young

    2004-05-01

    DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression in various types of model systems. Manufacturing high quality cDNA microarrays of microdeposition type depends on some key factors including a printing device, spotting pins, glass slides, spotting solution, and humidity during spotting. UsingEthe Microgrid II TAS model printing device, this study defined the optimal conditions for producing high density, high quality cDNA microarrays with the least amount of cDNA product. It was observed that aminosilane-modified slides were superior to other types of surface modified-slides. A humidity of 30+/-3% in a closed environment and the overnight drying of the spotted slides gave the best conditions for arraying. In addition, the cDNA dissolved in 30% DMSO gave the optimal conditions for spotting compared to the 1X ArrayIt, 3X SSC and 50% DMSO. Lastly, cDNA in the concentration range of 100-300 ng/ micro l was determined to be best for arraying and post-processing. Currently, the printing system in this study yields reproducible 9000 spots with a spot size 150 mm diameter, and a 200 nm spot spacing.

  9. Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication

    PubMed Central

    Egeland, Ryan D.; Southern, Edwin M.

    2005-01-01

    We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites. The acid is produced by electrochemical oxidation controlled by an array of independent microelectrodes. Deblocking is complete in a few seconds, when competing side-product reactions are minimal. We demonstrate the successful synthesis of 17mers and discrimination of single base pair mismatched hybrids. Features generated in this study are 40 μm wide, with sharply defined edges. The synthetic technique may be applicable to fabrication of other molecular arrays. PMID:16085751

  10. Surface ligation-based resonance light scattering analysis of methylated genomic DNA on a microarray platform.

    PubMed

    Ma, Lan; Lei, Zhen; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-05-10

    DNA methylation is a crucial epigenetic modification and is closely related to tumorigenesis. Herein, a surface ligation-based high throughput method combined with bisulfite treatment is developed for analysis of methylated genomic DNA. In this method, a DNA microarray is employed as a reaction platform, and resonance light scattering (RLS) of nanoparticles is used as the detection principle. The specificity stems from allele-specific ligation of Taq DNA ligase, which is further enhanced by improving the fidelity of Taq DNA ligase in a heterogeneous reaction. Two amplification techniques, rolling circle amplification (RCA) and silver enhancement, are employed after the ligation reaction and a gold nanoparticle (GNP) labeling procedure is used to amplify the signal. As little as 0.01% methylated DNA (i.e. 2 pmol L(-1)) can be distinguished from the cocktail of methylated and unmethylated DNA by the proposed method. More importantly, this method shows good accuracy and sensitivity in profiling the methylation level of genomic DNA of three selected colonic cancer cell lines. This strategy provides a high throughput alternative with reasonable sensitivity and resolution for cancer study and diagnosis.

  11. Phenotype microarray profiling of Zymomonas mobilis ZM4.

    PubMed

    Bochner, Barry; Gomez, Vanessa; Ziman, Michael; Yang, Shihui; Brown, Steven D

    2010-05-01

    In this study, we developed a Phenotype MicroArray (PM) protocol to profile cellular phenotypes in Zymomonas mobilis, which included a standard set of nearly 2,000 assays for carbon, nitrogen, phosphorus and sulfur source utilization, nutrient stimulation, pH and osmotic stresses, and chemical sensitivities with 240 inhibitory chemicals. We observed two positive assays for C-source utilization (fructose and glucose) using the PM screen, which uses redox chemistry and cell respiration as a universal reporter to profile growth phenotypes in a high-throughput 96-well plate-based format. For nitrogen metabolism, the bacterium showed a positive test results for ammonia, aspartate, asparagine, glutamate, glutamine, and peptides. Z. mobilis appeared to use a diverse array of P-sources with two exceptions being pyrophosphate and tripolyphosphate. The assays suggested that Z. mobilis uses both inorganic and organic compounds as S-sources. No stimulation by nutrients was detected; however, there was evidence of partial inhibition by purines and pyrimidines, NAD, and deferoxamine. Z. mobilis was relatively resistant to acid pH, tolerating a pH down to about 4.0. It also tolerated phosphate, sulfate, and nitrate, but was rather sensitive to chloride and nitrite. Z. mobilis showed resistance to a large number of diverse chemicals that inhibit most bacteria. The information from PM analysis provides an overview of Z. mobilis physiology and a foundation for future comparisons of other wild-type and mutant Z. mobilis strains.

  12. Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

    PubMed Central

    Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

    2009-01-01

    Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the

  13. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays.

    PubMed

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-11-30

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.

  14. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

    PubMed Central

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-01-01

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs. PMID:27916881

  15. Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.

    PubMed

    Viadas, C; Rodríguez, M C; García-Lobo, J M; Sangari, F J; López-Goñi, I

    2009-10-01

    The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.

  16. Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

    PubMed

    Kappelhoff, Reinhild; Puente, Xose S; Wilson, Claire H; Seth, Arun; López-Otín, Carlos; Overall, Christopher M

    2017-08-07

    The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4-20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  17. Forensic applications of DNA profiling.

    PubMed

    Chow, S T; Ng, T L; Chao, T C

    1996-01-01

    Deoxyribonucleic acid (DNA) profiling is a powerful forensic tool to identify and individualise biological evidence recovered at crime scenes. Its discriminating power and evidential value are exemplified by many actual forensic cases. Development of new DNA typing techniques, such as the amplification of fluorescently tagged short tandem repeats, would further enhance this capability in forensic investigation.

  18. Global gene expression analysis of two Streptococcus thermophilus bacteriophages using DNA microarray.

    PubMed

    Duplessis, Martin; Russell, W Michael; Romero, Dennis A; Moineau, Sylvain

    2005-09-30

    A custom microarray was developed to study the temporal gene expression of the two groups of phages infecting the Gram-positive lactic acid bacterium Streptococcus thermophilus. The complete genomic sequence of the virulent cos-type phage DT1 (34,815 bp) and the pac-type phage 2972 (34,704 bp) were used for the construction of the microarray. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32 and 37 min) during phage infection and an expression curve was determined for each gene. Each phage gene was then classified into one of the three traditional transcription classes and these data were used to generate the complete transcriptional map of DT1 and 2972. Phage DT1 possesses 18 early genes, 12 middle genes and 12 late-expressed genes whereas 2972 has 16 early, 11 middle and 14 late genes. The trends of the phage gene expression profiles were also confirmed by slot blot hybridizations. Significant differences were observed when comparing the transcriptional maps of DT1 and 2972 with those already available for the S. thermophilus phages Sfi19 and Sfi21. To our knowledge, this report presents the first complete transcription analysis of bacteriophages infecting Gram-positive bacteria using the DNA microarray technology.

  19. Microarray-based DNA methylation study of Ewing's sarcoma of the bone.

    PubMed

    Park, Hye-Rim; Jung, Woon-Won; Kim, Hyun-Sook; Park, Yong-Koo

    2014-10-01

    Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing's sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing's sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing's sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing's sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10, OSM, APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing's sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing's sarcoma.

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

    PubMed Central

    2011-01-01

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

  1. Model selection and efficiency testing for normalization of cDNA microarray data

    PubMed Central

    Futschik, Matthias; Crompton, Toni

    2004-01-01

    In this study we present two novel normalization schemes for cDNA microarrays. They are based on iterative local regression and optimization of model parameters by generalized cross-validation. Permutation tests assessing the efficiency of normalization demonstrated that the proposed schemes have an improved ability to remove systematic errors and to reduce variability in microarray data. The analysis also reveals that without parameter optimization local regression is frequently insufficient to remove systematic errors in microarray data. PMID:15287982

  2. Design of a combinatorial DNA microarray for protein-DNA interaction studies

    PubMed Central

    Mintseris, Julian; Eisen, Michael B

    2006-01-01

    Background Discovery of precise specificity of transcription factors is an important step on the way to understanding the complex mechanisms of gene regulation in eukaryotes. Recently, double-stranded protein-binding microarrays were developed as a potentially scalable approach to tackle transcription factor binding site identification. Results Here we present an algorithmic approach to experimental design of a microarray that allows for testing full specificity of a transcription factor binding to all possible DNA binding sites of a given length, with optimally efficient use of the array. This design is universal, works for any factor that binds a sequence motif and is not species-specific. Furthermore, simulation results show that data produced with the designed arrays is easier to analyze and would result in more precise identification of binding sites. Conclusion In this study, we present a design of a double stranded DNA microarray for protein-DNA interaction studies and show that our algorithm allows optimally efficient use of the arrays for this purpose. We believe such a design will prove useful for transcription factor binding site identification and other biological problems. PMID:17018151

  3. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

    PubMed

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

    2015-01-01

    The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

  4. Using DNA Microarrays to Detect Multiple Pathogen Threats in Water.

    SciTech Connect

    Straub, Tim M.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Call, Douglas R.; Chandler, Darrell P.

    2004-06-01

    Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

  5. A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

    PubMed

    Accogli, Gianluca; Desantis, Salvatore; Martino, Nicola Antonio; Dell'Aquila, Maria Elena; Gemeiner, Peter; Katrlík, Jaroslav

    2016-10-01

    The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

  6. Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation.

    PubMed

    Afshari, C A; Nuwaysir, E F; Barrett, J C

    1999-10-01

    One major challenge facing today's cancer researchers and toxicologists is the development of new approaches for the identification of carcinogens and other environmental hazards. Here, we describe the potential impact of emerging technologies for measuring gene expression profiles on carcinogen identification and on the general field of toxicology. An example of one of these technologies is the use of cDNA microarray chips. We provide an overview to the key questions that are confronting investigators charged with determining the relative safety of natural or synthetic chemicals to which humans are exposed, followed by a discussion of how cDNA microarray technology may be applied to these questions. Gene chip technology is still a relatively new technology, and only a handful of studies have demonstrated its utility. However, as the technical hurdles to development are passed, the use of this methodology in addressing the questions raised here will be critical to increase the sensitivity of detection of the potential toxic effects of environmental chemicals and to understand their risks to humans.

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

    PubMed

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

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  8. Microarray profiling of circular RNAs in human papillary thyroid carcinoma

    PubMed Central

    Peng, Nianchun; Shi, Lixin; Zhang, Qiao; Hu, Ying; Wang, Nanpeng; Ye, Hui

    2017-01-01

    Background Non-coding circular RNAs (circRNAs) have displayed dysregulated expression in several human cancers. Here, we profiled the circRNA expression of papillary thyroid carcinoma (PTC) tumors to improve our understanding of PTC pathogenesis. Methods Microarray profiling was performed on 18 thyroid samples, consisting of six PTC tumors, six matching contralateral normal samples, and six benign thyroid lesions. After low-intensity filtering, hierarchical clustering revealed the circRNA expression patterns. Statistical analysis followed by qRT-PCR validation identified the differential circRNAs. MicroRNA (miRNA) target prediction software identified putative miRNA response elements (MREs), which were used to construct a network map of circRNA-miRNA interactions for the differential circRNAs. Bioinformatics platforms predicted cancer-related circRNA-miRNA associations and putative downstream target genes, respectively. Results A total of 88 circRNAs and 10 circRNAs were significantly upregulated and downregulated, respectively, in PTC tumors relative to normal thyroid tissue, while 129 circRNAs and 226 circRNAs were significantly upregulated and downregulated, respectively, in PTC tumors relative to benign thyroid lesions. A total of 12 upregulated and four downregulated circRNAs were overlapping between the foregoing comparisons. One downregulated circRNA (hsa_circRNA_100395) showed interactive potential with two cancer-related miRNAs (miR-141-3p and miR-200a-3p). From this analysis, we identified several promising cancer-related genes that may be targets of the dysregulated hsa_circRNA_100395/miR-141-3p/miR-200a-3p axis in PTC tumors. Conclusions circRNA dysregulation may play a role in PTC pathogenesis, and several key circRNAs show promise as candidate biomarkers for PTC. The hsa_circRNA_100395/miR-141-3p/ miR-200a-3p axis may be involved in the pathogenesis of PTC. PMID:28288173

  9. Fabrication of DNA Microarrays on Polydopamine-Modified Gold Thin Films for SPR Imaging Measurements

    PubMed Central

    Wood, Jennifer B.; Szyndler, Megan W.; Halpern, Aaron R.; Cho, Kyunghee

    2013-01-01

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

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

    PubMed

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

    2013-08-27

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

  11. Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

    PubMed

    Hirasawa, Takashi; Yoshikawa, Katsunori; Nakakura, Yuki; Nagahisa, Keisuke; Furusawa, Chikara; Katakura, Yoshio; Shimizu, Hiroshi; Shioya, Suteaki

    2007-08-01

    During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.

  12. Altered expression of genes of the Bmp/Smad and Wnt/calcium signaling pathways in the cone-only Nrl-/- mouse retina, revealed by gene profiling using custom cDNA microarrays.

    PubMed

    Yu, Jindan; He, Shirley; Friedman, James S; Akimoto, Masayuki; Ghosh, Debashis; Mears, Alan J; Hicks, David; Swaroop, Anand

    2004-10-01

    Many mammalian retinas are rod-dominant, and hence our knowledge of cone photoreceptor biology is relatively limited. To gain insights into the molecular differences between rods and cones, we compared the gene expression profile of the rod-dominated retina of wild type mouse with that of the cone-only retina of Nrl(-/-) (Neural retina leucine zipper knockout) mouse. Our analysis, using custom microarrays of eye-expressed genes, provided equivalent data using either direct or reference-based experimental designs, confirmed differential expression of rod- and cone-specific genes in the Nrl(-/-) retina and identified novel genes that could serve as candidates for retinopathies or for functional studies. In addition, we detected altered expression of several genes that encode cell signaling or structural proteins. Prompted by these findings, additional real-time PCR analysis revealed that genes belonging to the Bmp/Smad and Wnt/Ca(2+) signaling pathways are expressed in the mature wild type retina and that their expression is significantly altered in the Nrl(-/-) retina. Chromatin immunoprecipitation analysis of adult retina identified Bmp4 and Smad4, which are down-regulated in the Nrl(-/-) retina, as possible direct transcriptional targets of Nrl. Consistent with these studies, Bmp4 and Smad4 are expressed in the mature rod photoreceptors of mouse retina. Modulation of Bmp4 and/or Smad4 by Nrl may provide a mechanism for integrating diverse cell signaling networks in rods. We hypothesize that Bmp/Smad and Wnt/Ca(2+) pathways participate in cell-cell communication in the mature retina, and expression changes observed in the Nrl(-/-) retina reflect their biased utilization in rod versus cone homeostasis.

  13. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  14. In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

    PubMed

    Saaem, Ishtiaq; Ma, Kuo-Sheng; Marchi, Alexandria N; LaBean, Thomas H; Tian, Jingdong

    2010-02-01

    Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

  15. DNA microarray technology in toxicogenomics of aquatic models: methods and applications.

    PubMed

    Ju, Zhenlin; Wells, Melissa C; Walter, Ronald B

    2007-02-01

    Toxicogenomics represents the merging of toxicology with genomics and bioinformatics to investigate biological functions of genome in response to environmental contaminants. Aquatic species have traditionally been used as models in toxicology to characterize the actions of environmental stresses. Recent completion of the DNA sequencing for several fish species has spurred the development of DNA microarrays allowing investigators access to toxicogenomic approaches. However, since microarray technology is thus far limited to only a few aquatic species and derivation of biological meaning from microarray data is highly dependent on statistical arguments, the full potential of microarray in aquatic species research has yet to be realized. Herein we review some of the issues related to construction, probe design, statistical and bioinformatical data analyses, and current applications of DNA microarrays. As a model a recently developed medaka (Oryzias latipes) oligonucleotide microarray was described to highlight some of the issues related to array technology and its application in aquatic species exposed to hypoxia. Although there are known non-biological variations present in microarray data, it remains unquestionable that array technology will have a great impact on aquatic toxicology. Microarray applications in aquatic toxicogenomics will range from the discovery of diagnostic biomarkers, to establishment of stress-specific signatures and molecular pathways hallmarking the adaptation to new environmental conditions.

  16. DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii

    DTIC Science & Technology

    2004-11-15

    DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii Chien-Chung Chao Rickettsiae Diseases...TITLE AND SUBTITLE DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii 5a. CONTRACT NUMBER 5b...ANSI Std Z39-18 Rickettsiae • Gram negative coccobacillary bacteria • Obligate intracellular organisms • Arthropod-borne • Cause febrile diseases (mild

  17. DNA Microarray Profiling of a Diverse Collection of Nosocomial Methicillin-Resistant Staphylococcus aureus Isolates Assigns the Majority to the Correct Sequence Type and Staphylococcal Cassette Chromosome mec (SCCmec) Type and Results in the Subsequent Identification and Characterization of Novel SCCmec-SCCM1 Composite Islands

    PubMed Central

    Brennan, Orla M.; Deasy, Emily C.; Rossney, Angela S.; Kinnevey, Peter M.; Ehricht, Ralf; Monecke, Stefan; Coleman, David C.

    2012-01-01

    One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCCM1 from ST8/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmec types and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate. PMID:22869569

  18. Development of a DNA microarray for species identification of quarantine aphids.

    PubMed

    Lee, Won Sun; Choi, Hwalran; Kang, Jinseok; Kim, Ji-Hoon; Lee, Si Hyeock; Lee, Seunghwan; Hwang, Seung Yong

    2013-12-01

    Aphid pests are being brought into Korea as a result of increased crop trading. Aphids exist on growth areas of plants, and thus plant growth is seriously affected by aphid pests. However, aphids are very small and have several sexual morphs and life stages, so it is difficult to identify species on the basis of morphological features. This problem was approached using DNA microarray technology. DNA targets of the cytochrome c oxidase subunit I gene were generated with a fluorescent dye-labelled primer and were hybridised onto a DNA microarray consisting of specific probes. After analysing the signal intensity of the specific probes, the unique patterns from the DNA microarray, consisting of 47 species-specific probes, were obtained to identify 23 aphid species. To confirm the accuracy of the developed DNA microarray, ten individual blind samples were used in blind trials, and the identifications were completely consistent with the sequencing data of all individual blind samples. A microarray has been developed to distinguish aphid species. DNA microarray technology provides a rapid, easy, cost-effective and accurate method for identifying aphid species for pest control management. © 2013 Society of Chemical Industry.

  19. Use of Microarray to Analyze Gene Expression Profiles of Acute Effects of Prochloraz on Fathead Minnows Pimephales promelas

    EPA Science Inventory

    Microarray technology is a powerful tool to investigate the gene expression profiles for thousands of genes simultaneously. In recent years, microarrays have been used to characterize environmental pollutants and identify molecular mode(s) of action of chemicals including endocri...

  20. Use of Microarray to Analyze Gene Expression Profiles of Acute Effects of Prochloraz on Fathead Minnows Pimephales promelas

    EPA Science Inventory

    Microarray technology is a powerful tool to investigate the gene expression profiles for thousands of genes simultaneously. In recent years, microarrays have been used to characterize environmental pollutants and identify molecular mode(s) of action of chemicals including endocri...

  1. Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring

    PubMed Central

    Chan, Mei-Lin; Jaramillo, Gerardo; Hristova, Krassimira R.; Horsley, David A.

    2010-01-01

    A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

  2. DNA nanostructure-based universal microarray platform for high-efficiency multiplex bioanalysis in biofluids.

    PubMed

    Li, Zhenhua; Zhao, Bin; Wang, Dongfang; Wen, Yanli; Liu, Gang; Dong, Haoqing; Song, Shiping; Fan, Chunhai

    2014-10-22

    Microarrays of biomolecules have greatly promoted the development of the fields of genomics, proteomics, and clinical assays because of their remarkably parallel and high-throughput assay capability. Immobilization strategies for biomolecules on a solid support surface play a crucial role in the fabrication of high-performance biological microarrays. In this study, rationally designed DNA tetrahedra carrying three amino groups and one single-stranded DNA extension were synthesized by the self-assembly of four oligonucleotides, followed by high-performance liquid chromatography purification. We fabricated DNA tetrahedron-based microarrays by covalently coupling the DNA tetrahedron onto glass substrates. After their biorecognition capability was evaluated, DNA tetrahedron microarrays were utilized for the analysis of different types of bioactive molecules. The gap hybridization strategy, the sandwich configuration, and the engineering aptamer strategy were employed for the assay of miRNA biomarkers, protein cancer biomarkers, and small molecules, respectively. The arrays showed good capability to anchor capture biomolecules for improving biorecognition. Addressable and high-throughput analysis with improved sensitivity and specificity had been achieved. The limit of detection for let-7a miRNA, prostate specific antigen, and cocaine were 10 fM, 40 pg/mL, and 100 nM, respectively. More importantly, we demonstrated that the microarray platform worked well with clinical serum samples and showed good relativity with conventional chemical luminescent immunoassay. We have developed a novel approach for the fabrication of DNA tetrahedron-based microarrays and a universal DNA tetrahedron-based microarray platform for the detection of different types of bioactive molecules. The microarray platform shows great potential for clinical diagnosis.

  3. DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases

    PubMed Central

    Cao, Boyang; Wang, Suwei; Tian, Zhenyang; Hu, Pinliang; Feng, Lu; Wang, Lei

    2015-01-01

    This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs. PMID:26208181

  4. DNA microarray technology for target identification and validation.

    PubMed

    Jayapal, Manikandan; Melendez, Alirio J

    2006-01-01

    1. Microarrays, a recent development, provide a revolutionary platform to analyse thousands of genes at once. They have enormous potential in the study of biological processes in health and disease and, perhaps, microarrays have become crucial tools in diagnostic applications and drug discovery. 2. Microarray based studies have provided the essential impetus for biomedical experiments, such as identification of disease-causing genes in malignancies and regulatory genes in the cell cycle mechanism. Microarrays can identify genes for new and unique potential drug targets, predict drug responsiveness for individual patients and, finally, initiate gene therapy and prevention strategies. 3. The present article reviews the principles and technological concerns, as well as the steps involved in obtaining and analysing of data. Furthermore, applications of microarray based experiments in drug target identifications and validation strategies are discussed. 4. To exemplify how this tool can be useful, in the present review we provide an overview of some of the past and potential future aspects of microarray technology and present a broad overview of this rapidly growing field.

  5. DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases.

    PubMed

    Cao, Boyang; Wang, Suwei; Tian, Zhenyang; Hu, Pinliang; Feng, Lu; Wang, Lei

    2015-01-01

    This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.

  6. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

    PubMed

    Hughes, T R; Mao, M; Jones, A R; Burchard, J; Marton, M J; Shannon, K W; Lefkowitz, S M; Ziman, M; Schelter, J M; Meyer, M R; Kobayashi, S; Davis, C; Dai, H; He, Y D; Stephaniants, S B; Cavet, G; Walker, W L; West, A; Coffey, E; Shoemaker, D D; Stoughton, R; Blanchard, A P; Friend, S H; Linsley, P S

    2001-04-01

    We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

  7. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.

    PubMed

    Nylund, Lotta; Heilig, Hans G H J; Salminen, Seppo; de Vos, Willem M; Satokari, Reetta

    2010-11-01

    The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Profiling glycosyltransferase activities by tritium imaging of glycan microarrays.

    PubMed

    Serna, Sonia; Hokke, Cornelis H; Weissenborn, Martin; Flitsch, Sabine; Martin-Lomas, Manuel; Reichardt, Niels-Christian

    2013-05-10

    High-throughput microarray technology has been combined with ultrasensitive and high-resolution tritium autoradiography to create a new platform for the quantitative detection of glycosyltransferase activity on glycan arrays. In addition, we show full compatibility with the use of fluorescently labeled lectins to help with the stereochemical assignment of newly formed glycoside linkages.

  9. Gene expression profiling in peanut using oligonucleotide microarrays

    USDA-ARS?s Scientific Manuscript database

    Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently have a moderately significant number of ESTs been released into the public domain. Utilization of these ESTs for the oligonucleotide microarrays provides a means to investigate l...

  10. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    PubMed Central

    Kim, Hye Young; Lee, Seo Eun; Kim, Min Jung; Han, Jin Il; Kim, Bo Kyung; Lee, Yong Sung; Lee, Young Seek; Kim, Jin Hyuk

    2007-01-01

    Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of spot images. Results We developed a governing equation of cDNA deposition during evaporation of a drop in the microarray spotting process. The governing equation included four parameters: the surface site density on the support, the extrapolated equilibrium constant for the binding of cDNA molecules with surface sites on glass slides, the macromolecular interaction factor, and the volume constant of a drop of cDNA solution. We simulated cDNA deposition from the single model equation by varying the value of the parameters. The morphology of the resulting cDNA deposit can be classified into three types: a doughnut shape, a peak shape, and a volcano shape. The spot morphology can be changed into a flat shape by varying the experimental conditions while considering the parameters of the governing equation of cDNA deposition. The four parameters were estimated by fitting the governing equation to the real microarray images. With the results of the simulation and the parameter estimation, the phenomenon of the formation of cDNA deposits in each type was investigated. Conclusion This study explains how various spot shapes can exist and suggests which parameters are to be adjusted for obtaining a good spot. This system is able to explore the cDNA microarray spotting process in a predictable, manageable and descriptive manner. We hope it can provide a way to predict the incidents that can occur during a real cDNA microarray experiment, and produce useful data for several research applications

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

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

  13. Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm

    PubMed Central

    Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

    2015-01-01

    DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively. PMID:26284175

  14. Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

    PubMed

    Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

    2015-01-01

    DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

  15. Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

    PubMed

    Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

    2011-06-01

    This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

  16. Determining genetic expression profiles in C. elegans using microarray and real-time PCR.

    PubMed

    Guthmueller, Kassandra L; Yoder, Maggie L; Holgado, Andrea M

    2011-07-30

    Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues (1) demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers (2) suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

  17. Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency

    PubMed Central

    Rosenberg, Jacob M.; Price, Jordan V.; Barcenas-Morales, Gabriela; Ceron-Gutierrez, Lourdes; Davies, Sophie; Kumararatne, Dinakantha S.; Döffinger, Rainer; Utz, Paul J.

    2015-01-01

    Background Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described. Objective We sought to profile ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assess the sensitivity and specificity of protein microarrays for ACAA identification and discovery. Methods Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 patients with immunodeficiency and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro. Results Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro. Conclusion Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency. PMID:26365387

  18. Automation of cDNA microarray hybridization and washing yields improved data quality.

    PubMed

    Yauk, Carole; Berndt, Lynn; Williams, Andrew; Douglas, George R

    2005-07-29

    Microarray technology allows the analysis of whole-genome transcription within a single hybridization, and has become a standard research tool. It is extremely important to minimize variation in order to obtain high quality microarray data that can be compared among experiments and laboratories. The majority of facilities implement manual hybridization approaches for microarray studies. We developed an automated method for cDNA microarray hybridization that uses equivalent pre-hybridization, hybridization and washing conditions to the suggested manual protocol. The automated method significantly decreased variability across microarray slides compared to manual hybridization. Although normalized signal intensities for buffer-only spots across the chips were identical, significantly reduced variation and inter-quartile ranges were obtained using the automated workstation. This decreased variation led to improved correlation among technical replicates across slides in both the Cy3 and Cy5 channels.

  19. Power of deep sequencing and agilent microarray for gene expression profiling study.

    PubMed

    Feng, Lin; Liu, Hang; Liu, Yu; Lu, Zhike; Guo, Guangwu; Guo, Suping; Zheng, Hongwei; Gao, Yanning; Cheng, Shujun; Wang, Jian; Zhang, Kaitai; Zhang, Yong

    2010-06-01

    Next-generation sequencing-based Digital Gene Expression tag profiling (DGE) has been used to study the changes in gene expression profiling. To compare the quality of the data generated by microarray and DGE, we examined the gene expression profiles of an in vitro cell model with these platforms. In this study, 17,362 and 15,938 genes were detected by microarray and DGE, respectively, with 13,221 overlapping genes. The correlation coefficients between the technical replicates were >0.99 and the detection variance was <9% for both platforms. The dynamic range of microarray was fixed with four orders of magnitude, whereas that of DGE was extendable. The consistency of the two platforms was high, especially for those abundant genes. It was more difficult for the microarray to distinguish the expression variation of less abundant genes. Although microarrays might be eventually replaced by DGE or transcriptome sequencing (RNA-seq) in the near future, microarrays are still stable, practical, and feasible, which may be useful for most biological researchers.

  20. Heat Survival and Phenotype Microarray Profiling of Salmonella Typhimurium Mutants.

    PubMed

    Dawoud, Turki M; Khatiwara, Anita; Park, Si Hong; Davis, Morgan L; Baker, Christopher A; Ricke, Steven C; Kwon, Young Min

    2017-02-01

    Contamination of food products by pathogenic microorganisms continues to be a major public health and food industry concern. Non-typhoidal Salmonella species have led to numerous outbreaks associated with various foods. A wide variety of methods have been applied and introduced for treatment of fresh foods to eliminate pathogenic as well as spoilage microorganisms. Salmonella can become exposed to elevated temperatures while associated with hosts such as poultry. In addition, heat treatment is also applied at various stages of processing to retain the shelf life of food products. Despite this, these microorganisms may overcome exposure to such treatments through the efficient expression of stress response mechanisms and result in illness following consumption. Thermal stress induces a range of destructive exposures to bacterial cells such as protein damage and DNA damage caused by reactive oxygen species. In this study, we chose three genes (∆recD, ∆STM14_5307, and ∆aroD) associated with conditionally essential genes required for different aspects of optimal growth at 42 °C and evaluated the responses of wild type and mutant Salmonella Typhimurium strains to uncover potential mechanisms that may enable survival and resistance under thermal stress. The RecBCD complex that initiates repair of double-stranded DNA breaks through homologous recombination. STM14_5307 is a transcriptional regulator involved in stationary phase growth and inositol metabolism. The gene aroD is involved in metabolism and stationary phase growth. These strains were characterized via high throughput phenotypic profiling in response to two different growth temperatures (37 °C (human host temperature) and 42 °C (poultry host temperature)). The ∆aroD strain exhibited the highest sensitivity to the various temperatures followed by the ∆recD and ∆STM14_5307 strains, respectively. Achieving more understanding of the molecular mechanisms of heat survival may lead to the development

  1. Biostatistical Considerations of the Use of Genomic DNA Reference in Microarrays

    SciTech Connect

    Yang, Yunfeng; Zhu, Mengxia; Wu, Liyou; Zhou, Jizhong

    2008-01-01

    Using genomic DNA as common reference in microarray experiments has recently been tested by different laboratories (2, 3, 5, 7, 9, 20, 24-26). While some reported that experimental results of microarrays using genomic DNA reference conforms nicely to those obtained by cDNA: cDNA co-hybridization method, others acquired poor results. We hypothesize that these conflicting reports could be resolved by biostatistical analyses. To test it, microarray experiments were performed in a -proteobacterium Shewanella oneidensis. Pair-wise comparison of three experimental conditions was obtained either by direct cDNA: cDNA co-hybridization, or by indirect calculation through a Shewanella genomic DNA reference. Several major biostatistical techniques were exploited to reduce the amount of inconsistency between both methods and the results were assessed. We discovered that imposing the constraint of minimal number of replicates, logarithmic transformation and random error analyses could significantly improve the data quality. Therefore, it is useful to adopt these biostatistical techniques for microarray data analysis using genomic DNA as reference.

  2. FPGA based system for automatic cDNA microarray image processing.

    PubMed

    Belean, Bogdan; Borda, Monica; Le Gal, Bertrand; Terebes, Romulus

    2012-07-01

    Automation is an open subject in DNA microarray image processing, aiming reliable gene expression estimation. The paper presents a novel shock filter based approach for automatic microarray grid alignment. The proposed method brings up significantly reduced computational complexity compared to state of the art approaches, while similar results in terms of accuracy are achieved. Based on this approach, we also propose an FPGA based system for microarray image analysis that eliminates the shortcomings of existing software platforms: user intervention, increased computational time and cost. Our system includes application-specific architectures which involve algorithm parallelization, aiming fast and automated cDNA microarray image processing. The proposed automated image processing chain is implemented both on a general purpose processor and using the developed hardware architectures as co-processors in a FPGA based system. The comparative results included in the last section show that an important gain in terms of computational time is obtained using hardware based implementations.

  3. [DNA microarrays--perspective of application for drug effectivity and safety evaluation].

    PubMed

    Roman, Iza

    2008-01-01

    Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). Microarray has been successfully applied to the high throughput simultaneous expression of many thousands of genes in a single experiment. One important application of DNA microarray technology, within the context of drugs effectiveness and safety evaluation studies, is its use as a screening tool for the identification of biochemical pathways, potential targets for novel molecular therapeutics, for the identification of molecular mechanisms of toxicity and to understand and predict individual drug sensitivity and resistance. The purpose of this review is presentation of the utility of DNA microarray technology in all phases of the drug discovery process.

  4. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    PubMed Central

    Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

    2002-01-01

    Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

  5. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis.

    PubMed

    Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

    2002-10-30

    T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

  6. An Undergraduate Laboratory Exercise to Study the Effect of Darkness on Plant Gene Expression Using DNA Microarray

    ERIC Educational Resources Information Center

    Chang, Ming-Mei; Briggs, George M.

    2007-01-01

    DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory…

  7. An Undergraduate Laboratory Exercise to Study the Effect of Darkness on Plant Gene Expression Using DNA Microarray

    ERIC Educational Resources Information Center

    Chang, Ming-Mei; Briggs, George M.

    2007-01-01

    DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory…

  8. Novel multistranded, alternative, plasmid and helical transitional DNA and RNA microarrays: implications for therapeutics.

    PubMed

    Gagna, Claude E; Lambert, W Clark

    2009-05-01

    Novel multistranded and alternative DNA, RNA and plasmid microarrays (transitional structural nucleic acid microarrays) have been developed that allows for the immobilization of intact, nondenatured, double-stranded DNA, double-stranded RNA, and alternative and multistranded nucleic acids. It also allows for the study of transitional changes that occur in the structure of DNA and RNA. Alternative types of DNA, RNA and multistranded nucleic acids are immobilized by a variety of different surface chemistries (i.e., noncovalent or covalent) onto a novel substrate surface. This technology represents the next generation of microarrays, which will aid in the characterization of nucleic acid structure and function, and accelerate the discovery of drugs that bind to nucleic acids. In addition, we demonstrate four novel techniques that are the first practical applications of the microarray, that is, transitional structural chemogenomics, transitional structural chemoproteomics, transitional structural pharmacogenomics and transitional structural pharmacoproteomics. These novel nucleic acid microarrays, together with pharmacogenomics, can be used to improve the study of DNA and RNA structure, gene expression, drug development and treatment of various diseases.

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

    PubMed

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

    2016-07-15

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

  10. Three microarray platforms: an analysis of their concordance in profiling gene expression

    PubMed Central

    Petersen, David; Chandramouli, GVR; Geoghegan, Joel; Hilburn, Joanne; Paarlberg, Jonathon; Kim, Chang Hee; Munroe, David; Gangi, Lisa; Han, Jing; Puri, Raj; Staudt, Lou; Weinstein, John; Barrett, J Carl; Green, Jeffrey; Kawasaki, Ernest S

    2005-01-01

    Background Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25–30 base), long oligonucleotide (50–80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. Results The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%. Conclusion Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change. PMID:15876355

  11. Multivariate analysis of low-dose radiation-associated changes in cytokine gene expression profiles using microarray technology.

    PubMed

    Albanese, Joseph; Martens, Kelly; Karanitsa, Leonid V; Karkanitsa, Leonid V; Schreyer, Suzanne K; Dainiak, Nicholas

    2007-04-01

    The availability of microarray technology, which permits evaluation of the entire cellular transcriptome in a single experiment, has provided new insights on the function of the genome under normal and pathological conditions, as well as in response to genotoxic stimuli, including ionizing radiation. The aims of this study were to: 1) determine whether specific cytokine gene expression profiles can be delineated in individuals exposed to chronic, low-dose radiation; and 2) compare analyses from three multivariate analytic methodologies, hierarchical clustering analysis (HCA), principal component analysis (PCA), and projection pursuit (PP), in evaluating transcriptional responses in human mononuclear cells to low doses of ionizing radiation (IR), as determined by cDNA microarrays. Total RNA isolated from mononuclear cells of 19 apparently healthy adult individuals exposed to low doses of IR ranging from 0.18 mSv to 49 mSv over a period of 11 to 13 years, as a result of the Chernobyl Nuclear Power Plant catastrophe, was reverse transcribed in the presence of radioactive dATP to generate radiolabeled complementary cDNA. Target cDNA was hybridized to human cytokine and receptor arrays and mRNA transcriptional patterns were evaluated using HCA, PCA, and PP. Statistical analyses of the data generated from 19 microarrays revealed distinct gene expression patterns in mononuclear cells of individuals exposed to radiation doses of greater than 10 mSv or less than 10 mSv. Genes encompassed within clusters discerned by HCA, PCA, and PP varied depending on the methodology used to analyze the microarray data. The most frequently expressed genes across all radiation doses were serine/threonine protein kinase receptor (11/19), transforming growth factor (TGF) receptor (11/19), EB13 (10/19), and CD40 ligand. Although our findings suggest that it may be possible to assign gene expression profiles to low-dose-irradiated individuals, we show that gene expression profiles vary

  12. The emergence and diffusion of DNA microarray technology

    PubMed Central

    Lenoir, Tim; Giannella, Eric

    2006-01-01

    The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. We discuss the role of federal funding of academic research in the microarray field, and the multiple pathways through which federally supported development of commercial microarray technologies have transformed core academic research fields. Our study confirms the picture put forward by several scholars that the open character of networked economies is what makes them truly innovative. In an open system innovations emerge from the network. The emergence and diffusion of microarray technologies we have traced here provides an excellent example of an open system of innovation in action. Whether they originated in a startup company environment that operated like a think-tank, such as Affymax, the research labs of a large firm, such as Agilent, or within a research university, the inventors we have followed drew heavily on knowledge resources from all parts of the network in bringing microarray platforms to light. Federal funding for high-tech startups and new industrial development was important at several phases in the early history of microarrays, and federal funding of academic researchers using microarrays was fundamental

  13. Functional genomics in chickens: development of integrated-systems microarrays for transcriptional profiling and discovery of regulatory pathways.

    PubMed

    Cogburn, L A; Wang, X; Carre, W; Rejto, L; Aggrey, S E; Duclos, M J; Simon, J; Porter, T E

    2004-01-01

    The genetic networks that govern the differentiation and growth of major tissues of economic importance in the chicken are largely unknown. Under a functional genomics project, our consortium has generated 30 609 expressed sequence tags (ESTs) and developed several chicken DNA microarrays, which represent the Chicken Metabolic/Somatic (10 K) and Neuroendocrine/Reproductive (8 K) Systems (http://udgenome.ags.udel.edu/cogburn/). One of the major challenges facing functional genomics is the development of mathematical models to reconstruct functional gene networks and regulatory pathways from vast volumes of microarray data. In initial studies with liver-specific microarrays (3.1 K), we have examined gene expression profiles in liver during the peri-hatch transition and during a strong metabolic perturbation-fasting and re-feeding-in divergently selected broiler chickens (fast vs. slow-growth lines). The expression of many genes controlling metabolic pathways is dramatically altered by these perturbations. Our analysis has revealed a large number of clusters of functionally related genes (mainly metabolic enzymes and transcription factors) that control major metabolic pathways. Currently, we are conducting transcriptional profiling studies of multiple tissues during development of two sets of divergently selected broiler chickens (fast vs. slow growing and fat vs. lean lines). Transcriptional profiling across multiple tissues should permit construction of a detailed genetic blueprint that illustrates the developmental events and hierarchy of genes that govern growth and development of chickens. This review will briefly describe the recent acquisition of chicken genomic resources (ESTs and microarrays) and our consortium's efforts to help launch the new era of functional genomics in the chicken.

  14. Expression profile of long non-coding RNAs in colorectal cancer: A microarray analysis.

    PubMed

    Luo, Jia; Xu, Luning; Jiang, Yigui; Zhuo, Dexiang; Zhang, Shengjun; Wu, Lianhui; Xu, Huadong; Huang, Yue

    2016-04-01

    Colorectal cancer (CRC) is one of the most prevalent malignant tumors and the second cause of cancer-related mortality worldwide. Due to increased morbidity and mortality rates, there is an urgent need to understand the pathogenesis of CRC, discover strategies that can improve diagnosis, and ultimately identify therapies targeting this disease. Over the past several years, research into tumor progression mechanisms has been devoted to identifying and understanding various coding and non-coding regions of the genome and how these genetic variants may affect tumorigenesis and progression. Recently, long non-coding RNAs (lncRNAs), which are non‑protein coding transcripts longer than 200 nucleotides, have emerged as a key aspect in tumor pathogenesis. In the present study, we examined the lncRNA and mRNA expression profiles in 4 patients with colon adenocarcinoma, with paired adjacent normal tissues as controls. Microarray data showed that a total of 3,523 lncRNAs and 2,515 mRNAs were consistently differentially expressed in the CRC tissues compared to adjacent normal tissues. Upon comparison of the differentially expressed transcripts between the groups, we identified 22 pathways which were related to the upregulated transcripts and 24 pathways that corresponded to the downregulated transcripts. Gene ontology analysis revealed that the upregulated transcripts were predominantly enriched in DNA metabolic processes, and the downregulated transcripts were predominantly enriched in organic hydroxyl compound metabolic processes. Coding-non-coding gene co-expression analysis showed that these differentially expressed lncRNAs were closely correlated with 'Wnt signaling pathway' components, whose aberrant activation plays a central role in CRC, indicating that a functional correlation exists between them. In conclusion, the results of the microarray and informatic analysis strongly suggest that lncRNA dysregulation is involved in the complicated process of CRC development

  15. Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

    PubMed

    Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

    2014-10-01

    Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

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

    SciTech Connect

    Sadikovic, Bekim; Andrews, Joseph; Rodenhiser, David I.

    2007-12-15

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

  17. Methods in DNA methylation profiling

    PubMed Central

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Huey-Jen L; Huang, Tim H-M

    2010-01-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling. PMID:20526417

  18. Methods in DNA methylation profiling.

    PubMed

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Juey-Jen L; Huang, Tim H-M

    2009-12-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.

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

    PubMed

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

    2006-11-01

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

  20. Application of L-cystein derivative to DNA microarray.

    PubMed

    Nakauchi, Gen; Inaki, Yoshiaki; Kitaoka, Shiho; Yokoyama, Chieko; Tanabe, Tadashi

    2002-01-01

    S-carboxymethyl-L-cystein derivatives of nucleic acid bases were prepared as DNA chip probe. These compounds in vitro have been found to form stable complex with oligo-DNA and RNA. This paper deals with preparing new DNA chip using L-cystein derivative synthetic nucleotides as probe and immobilized it to quartz plate by photosensitive PVA. Then the chip exposed with FITC labeled target DNA was observed by confocal fluorescence microscope.

  1. RNA-seq and microarray complement each other in transcriptome profiling

    PubMed Central

    2012-01-01

    Background RNA-seq and microarray are the two popular methods employed for genome-wide transcriptome profiling. Current comparison studies have shown that transcriptome quantified by these two methods correlated well. However, none of them have addressed if they complement each other, considering the strengths and the limitations inherent with them. The pivotal requirement to address this question is the knowledge of a well known data set. In this regard, HrpX regulome from pathogenic bacteria serves as an ideal choice as the target genes of HrpX transcription factor are well studied due to their central role in pathogenicity. Results We compared the performance of RNA-seq and microarray in their ability to detect known HrpX target genes by profiling the transcriptome from the wild-type and the hrpX mutant strains of γ-Proteobacterium Xanthomonas citri subsp. citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, rs > 0.76). Further, the expression levels quantified by RNA-seq and microarray for the significantly differentially expressed genes (DEGs) also well-correlated with qRT-PCR based quantification (rs = 0.58 to 0.94). Finally, in addition to the 55 newly identified DEGs, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods. Conclusions This study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. RNA-seq and microarray together provide a more comprehensive picture of HrpX regulome by uniquely identifying new DEGs. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling. PMID:23153100

  2. Role of DNA profiling in forensic odontology.

    PubMed

    Sakari, S Leena; Jimson, Sudha; Masthan, K M K; Jacobina, Jenita

    2015-04-01

    The recent advances in DNA profiling have made DNA evidence to be more widely accepted in courts. This has revolutionized the aspect of forensic odontology. DNA profiling/DNA fingerprinting has come a long way from the conventional fingerprints. DNA that is responsible for all the cell's activities, yields valuable information both in the healthy and diseased individuals. When other means of traditional identification become impossible following mass calamities or fire explosions, teeth provide a rich source of DNA as they have a high chemical as well as physical resistance. The recent evolution in the isolation of DNA and the ways of running a DNA fingerprint are highlighted in this literature review.

  3. Stability of double-stranded oligonucleotide DNA with a bulged loop: a microarray study

    PubMed Central

    2011-01-01

    Background DNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays. Results We synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account. Conclusions We show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well. PMID

  4. Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

    PubMed Central

    Zasedateleva, Olga A.; Mikheikin, Andrey L.; Turygin, Alexander Y.; Prokopenko, Dmitry V.; Chudinov, Alexander V.; Belobritskaya, Elena E.; Chechetkin, Vladimir R.; Zasedatelev, Alexander S.

    2008-01-01

    Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N1N2N3N4N5N6{N}-3′ consisted of a fixed hexamer motif N1N2N3N4N5N6 in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5′-NNG(A/T/C)GNN-3′ with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases. PMID:18474529

  5. Evaluation of high-resolution microarray platforms for genomic profiling of bone tumours

    PubMed Central

    2010-01-01

    Background Several high-density oligonucleotide microarray platforms are available for genome-wide single nucleotide polymorphism (SNP) detection and microarray-based comparative genomic hybridisation (array CGH), which may be used to detect copy number aberrations in human tumours. As part of the EuroBoNeT network of excellence for research on bone tumours (eurobonet.eu), we have evaluated four different commercial high-resolution microarray platforms in order to identify the most appropriate technology for mapping DNA copy number aberrations in such tumours. Findings DNA from two different cytogenetically well-characterized bone sarcoma cell lines, representing a simple and a complex karyotype, respectively, was tested in duplicate on four high-resolution microarray platforms; Affymetrix Genome-Wide Human SNP Array 6.0, Agilent Human Genome CGH 244A, Illumina HumanExon510s-duo and Nimblegen HG18 CGH 385 k WG tiling v1.0. The data was analysed using the platform-specific analysis software, as well as a platform-independent analysis algorithm. DNA copy number was measured at six specific chromosomes or chromosomal regions, and compared with the expected ratio based on available cytogenetic information. All platforms performed well in terms of reproducibility and were able to delimit and score small amplifications and deletions at similar resolution, but Agilent microarrays showed better linearity and dynamic range. The platform-specific analysis software provided with each platform identified in general correct copy numbers, whereas using a platform-independent analysis algorithm, correct copy numbers were determined mainly for Agilent and Affymetrix microarrays. Conclusions All platforms performed reasonably well, but Agilent microarrays showed better dynamic range, and like Affymetrix microarrays performed well with the platform-independent analysis software, implying more robust data. Bone tumours like osteosarcomas are heterogeneous tumours with complex

  6. DNA profiling of trace DNA recovered from bedding.

    PubMed

    Petricevic, Susan F; Bright, Jo-Anne; Cockerton, Sarah L

    2006-05-25

    Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.

  7. Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.

    PubMed

    Stevens-Kroef, Marian Jpl; van den Berg, Eva; Olde Weghuis, Daniel; Geurts van Kessel, Ad; Pfundt, Rolph; Linssen-Wiersma, Matty; Benjamins, Marloes; Dijkhuizen, Trijnie; Groenen, Patricia Jta; Simons, Annet

    2014-01-09

    Characteristic genomic abnormalities in patients with B cell chronic lymphocytic leukemia (CLL) have been shown to provide important prognostic information. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA), currently used in clinical diagnostics of CLL, are targeted tests aimed at specific genomic loci. Microarray-based genomic profiling is a new high-resolution tool that enables genome-wide analyses. The aim of this study was to compare two recently launched genomic microarray platforms, i.e., the CytoScan HD Array (Affymetrix) and the HumanOmniExpress Array (Illumina), with FISH and MLPA to ascertain whether these latter tests can be replaced by either one of the microarray platforms in a clinical diagnostic setting. Microarray-based genomic profiling and FISH were performed in all 28 CLL patients. For an unbiased comparison of the performance of both microarray platforms 9 patients were evaluated on both platforms, resulting in the identification of exactly identical genomic aberrations. To evaluate the detection limit of the microarray platforms we included 7 patients in which the genomic abnormalities were present in a relatively low percentage of the cells (range 5-28%) as previously determined by FISH. We found that both microarray platforms allowed the detection of copy number abnormalities present in as few as 16% of the cells. In addition, we found that microarray-based genomic profiling allowed the identification of genomic abnormalities that could not be detected by FISH and/or MLPA, including a focal TP53 loss and copy neutral losses of heterozygosity of chromosome 17p. From our results we conclude that although the microarray platforms exhibit a somewhat lower limit of detection compared to FISH, they still allow the detection of copy number abnormalities present in as few as 16% of the cells. By applying similar interpretation criteria, the results obtained from both platforms were comparable. In

  8. Sequential interim analyses of survival data in DNA microarray experiments

    PubMed Central

    2011-01-01

    Background Discovery of biomarkers that are correlated with therapy response and thus with survival is an important goal of medical research on severe diseases, e.g. cancer. Frequently, microarray studies are performed to identify genes of which the expression levels in pretherapeutic tissue samples are correlated to survival times of patients. Typically, such a study can take several years until the full planned sample size is available. Therefore, interim analyses are desirable, offering the possibility of stopping the study earlier, or of performing additional laboratory experiments to validate the role of the detected genes. While many methods correcting the multiple testing bias introduced by interim analyses have been proposed for studies of one single feature, there are still open questions about interim analyses of multiple features, particularly of high-dimensional microarray data, where the number of features clearly exceeds the number of samples. Therefore, we examine false discovery rates and power rates in microarray experiments performed during interim analyses of survival studies. In addition, the early stopping based on interim results of such studies is evaluated. As stop criterion we employ the achieved average power rate, i.e. the proportion of detected true positives, for which a new estimator is derived and compared to existing estimators. Results In a simulation study, pre-specified levels of the false discovery rate are maintained in each interim analysis, where reduced levels as used in classical group sequential designs of one single feature are not necessary. Average power rates increase with each interim analysis, and many studies can be stopped prior to their planned end when a certain pre-specified power rate is achieved. The new estimator for the power rate slightly deviates from the true power rate but is comparable to other estimators. Conclusions Interim analyses of microarray experiments can provide evidence for early stopping of

  9. ArrayQuest: a web resource for the analysis of DNA microarray data

    PubMed Central

    Argraves, Gary L; Jani, Saurin; Barth, Jeremy L; Argraves, W Scott

    2005-01-01

    Background Numerous microarray analysis programs have been created through the efforts of Open Source software development projects. Providing browser-based interfaces that allow these programs to be executed over the Internet enhances the applicability and utility of these analytic software tools. Results Here we present ArrayQuest, a web-based DNA microarray analysis process controller. Key features of ArrayQuest are that (1) it is capable of executing numerous analysis programs such as those written in R, BioPerl and C++; (2) new analysis programs can be added to ArrayQuest Methods Library at the request of users or developers; (3) input DNA microarray data can be selected from public databases (i.e., the Medical University of South Carolina (MUSC) DNA Microarray Database or Gene Expression Omnibus (GEO)) or it can be uploaded to the ArrayQuest center-point web server into a password-protected area; and (4) analysis jobs are distributed across computers configured in a backend cluster. To demonstrate the utility of ArrayQuest we have populated the methods library with methods for analysis of Affymetrix DNA microarray data. Conclusion ArrayQuest enables browser-based implementation of DNA microarray data analysis programs that can be executed on a Linux-based platform. Importantly, ArrayQuest is a platform that will facilitate the distribution and implementation of new analysis algorithms and is therefore of use to both developers of analysis applications as well as users. ArrayQuest is freely available for use at . PMID:16321157

  10. ArrayQuest: a web resource for the analysis of DNA microarray data.

    PubMed

    Argraves, Gary L; Jani, Saurin; Barth, Jeremy L; Argraves, W Scott

    2005-12-01

    Numerous microarray analysis programs have been created through the efforts of Open Source software development projects. Providing browser-based interfaces that allow these programs to be executed over the Internet enhances the applicability and utility of these analytic software tools. Here we present ArrayQuest, a web-based DNA microarray analysis process controller. Key features of ArrayQuest are that (1) it is capable of executing numerous analysis programs such as those written in R, BioPerl and C++; (2) new analysis programs can be added to ArrayQuest Methods Library at the request of users or developers; (3) input DNA microarray data can be selected from public databases (i.e., the Medical University of South Carolina (MUSC) DNA Microarray Database or Gene Expression Omnibus (GEO)) or it can be uploaded to the ArrayQuest center-point web server into a password-protected area; and (4) analysis jobs are distributed across computers configured in a backend cluster. To demonstrate the utility of ArrayQuest we have populated the methods library with methods for analysis of Affymetrix DNA microarray data. ArrayQuest enables browser-based implementation of DNA microarray data analysis programs that can be executed on a Linux-based platform. Importantly, ArrayQuest is a platform that will facilitate the distribution and implementation of new analysis algorithms and is therefore of use to both developers of analysis applications as well as users. ArrayQuest is freely available for use at http://proteogenomics.musc.edu/arrayquest.html.

  11. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    PubMed

    Liang, Lifeng; Wang, Cassie T; Sun, Xiaofang; Liu, Lian; Li, Man; Witz, Craig; Williams, Daniel; Griffith, Jason; Skorupski, Josh; Haddad, Ghassan; Gill, Jimmy; Wang, Wei-Hua

    2013-01-01

    A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.

  12. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  13. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  14. A versatile protein microarray platform enabling antibody profiling against denatured proteins.

    PubMed

    Wang, Jie; Barker, Kristi; Steel, Jason; Park, Jin; Saul, Justin; Festa, Fernanda; Wallstrom, Garrick; Yu, Xiaobo; Bian, Xiaofang; Anderson, Karen S; Figueroa, Jonine D; LaBaer, Joshua; Qiu, Ji

    2013-06-01

    We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection. We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation (IVTT) system. Escherichia coli lysates were added to the plasma blocking buffer to reduce nonspecific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment. We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins. This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2016-01-01

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

  16. Profiling human serum antibodies with a carbohydrate antigen microarray

    PubMed Central

    Oyelaran, Oyindasola; McShane, Lisa M.; Dodd, Lori; Gildersleeve, Jeffrey C.

    2009-01-01

    Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, inter-individual biological variability, and intra-individual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling. PMID:19624168

  17. Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays.

    PubMed

    Pingel, Julia; Buhot, Arnaud; Calemczuk, Roberto; Livache, Thierry

    2012-01-15

    The possibility to detect low abundant DNA point mutations is essential for early cancer diagnosis and/or prognosis. Furthermore, in order to be less invasive, the somatic mutations are not only sought in tumor extract samples but also from body fluids or stools rendering their content even more diluted compared to the wild type sequences. In this short communication, we propose two protocols based on temperature scans or cycles for the enrichment of the mutation strands hybridized on microarrays. We predict numerically and confirm experimentally a 10-fold increase in the fraction of mutated DNA hybridized on the microarray compared to the sample content. Coupled to more standard solution phase enrichment techniques, it would be possible to lower by one order of magnitude the current detection limit with the advantage of multiple mutation detections offered by the microarray technology. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. A 7872 cDNA microarray and its use in bovine functional genomics.

    PubMed

    Everts, Robin E; Band, Mark R; Liu, Z Lewis; Kumar, Charu G; Liu, Lei; Loor, Juan J; Oliveira, Rosane; Lewin, Harris A

    2005-05-15

    The strategy used to create and annotate a 7872 cDNA microarray from cattle placenta and spleen cDNA sequences is described. This microarray contains approximately 6300 unique genes, as determined by BLASTN and TBLASTX similarity search against the human and mouse UniGene and draft human genome sequence databases (build 34). Sequences on the array were annotated with gene ontology (GO) terms, thereby facilitating data analysis and interpretation. A total of 3244 genes were annotated with GO terms. The array is rich in sequences encoding transcription factors, signal transducers and cell cycle regulators. Current research being conducted with this array is described, and an overview of planned improvements in our microarray platform for cattle functional genomics is presented.

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

    PubMed Central

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  20. A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

    PubMed

    Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-04-25

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

  1. Assessment and integration of publicly available SAGE, cDNA microarray, and oligonucleotide microarray expression data for global coexpression analyses.

    PubMed

    Griffith, Obi L; Pleasance, Erin D; Fulton, Debra L; Oveisi, Mehrdad; Ester, Martin; Siddiqui, Asim S; Jones, Steven J M

    2005-10-01

    Large amounts of gene expression data from several different technologies are becoming available to the scientific community. A common practice is to use these data to calculate global gene coexpression for validation or integration of other "omic" data. To assess the utility of publicly available datasets for this purpose we have analyzed Homo sapiens data from 1202 cDNA microarray experiments, 242 SAGE libraries, and 667 Affymetrix oligonucleotide microarray experiments. The three datasets compared demonstrate significant but low levels of global concordance (rc<0.11). Assessment against Gene Ontology (GO) revealed that all three platforms identify more coexpressed gene pairs with common biological processes than expected by chance. As the Pearson correlation for a gene pair increased it was more likely to be confirmed by GO. The Affymetrix dataset performed best individually with gene pairs of correlation 0.9-1.0 confirmed by GO in 74% of cases. However, in all cases, gene pairs confirmed by multiple platforms were more likely to be confirmed by GO. We show that combining results from different expression platforms increases reliability of coexpression. A comparison with other recently published coexpression studies found similar results in terms of performance against GO but with each method producing distinctly different gene pair lists.

  2. Optimal cDNA microarray design using expressed sequence tags for organisms with limited genomic information

    PubMed Central

    Chen, Yian A; Mckillen, David J; Wu, Shuyuan; Jenny, Matthew J; Chapman, Robert; Gross, Paul S; Warr, Gregory W; Almeida, Jonas S

    2004-01-01

    Background Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes. Results Sequence similarity between ESTs, quantified by the E-value of pair-wise alignment, was used as a surrogate for expected hybridization between corresponding sequences. Using this value as a measure of dissimilarity, sequence redundancy reduction was performed by hierarchical cluster analyses. The choice of how many microarray probes to retain was made based on an index developed for this research: a sequence diversity index (SDI) within a sequence diversity plot (SDP). This index tracked the decreasing within-cluster sequence diversity as the number of clusters increased. For a given stage in the agglomeration procedure, the EST having the highest similarity to all the other sequences within each cluster, the centroid EST, was selected as a microarray probe. A small dataset of ESTs from Atlantic white shrimp (Litopenaeus setiferus) was used to test this algorithm so that the detailed results could be examined. The functional representative level of the selected probes was quantified using Gene Ontology (GO) annotations. Conclusions For organisms with limited genomic information, combining hierarchical clustering methods to analyze ESTs can yield an optimal cDNA microarray design. If

  3. Electronic hybridization detection in microarray format and DNA genotyping

    PubMed Central

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-01-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

  4. Electronic hybridization detection in microarray format and DNA genotyping

    NASA Astrophysics Data System (ADS)

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-02-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

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

    USDA-ARS?s Scientific Manuscript database

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

  6. USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    EPA Science Inventory

    USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
    IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
    Reproductive Toxicology Divisio...

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

    PubMed

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

    2012-01-01

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

  8. Direct identification of chlamydiae from clinical samples using a DNA microarray assay: a validation study.

    PubMed

    Borel, Nicole; Kempf, Evelyne; Hotzel, Helmut; Schubert, Evelyn; Torgerson, Paul; Slickers, Peter; Ehricht, Ralf; Tasara, Taurai; Pospischil, Andreas; Sachse, Konrad

    2008-02-01

    While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

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

    PubMed Central

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

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  10. USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    EPA Science Inventory

    USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
    IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
    Reproductive Toxicology Divisio...

  11. Comparison of DNA ploidy status and DNA ploidy-related parameters in malignant melanoma tissue microarrays and full sections.

    PubMed

    Korabiowska, Monika; Cordon-Cardo, Carlos; Buschmann, Nadine; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich

    2004-07-01

    A new high-throughput tissue-arraying technique, now frequently used in tumor pathology, requires standardization of methods of DNA analysis, previously applied in full histological sections. The main objectives of this study were to evaluate DNA ploidy status and DNA ploidy-related parameters using the CAS200 image analyzer in malignant melanoma tissue microarrays and to compare them with full histological sections. Comparison of DNA ploidy-related parameters, including percentage of diploid cells, percentage of aneuploid cells between 2c and 4c, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, percentage of octaploid cells, percentage of 16-ploid cells, and 5c exceeding rate, did not reveal any significant differences between malignant melanoma tissue microarrays and full sections. The DNA ploidy status according to Auer differed in 1 out of 59 cases investigated. Our study demonstrated that it is possible to evaluate DNA ploidy status and DNA ploidy-related parameters in tissue microarrays, which is of practical relevance to tumor pathology.

  12. Robust embryo identification using first polar body single nucleotide polymorphism microarray-based DNA fingerprinting.

    PubMed

    Treff, Nathan R; Su, Jing; Kasabwala, Natasha; Tao, Xin; Miller, Kathleen A; Scott, Richard T

    2010-05-01

    This study sought to validate a novel, minimally invasive system for embryo tracking by single nucleotide polymorphism microarray-based DNA fingerprinting of the first polar body. First polar body-based assignments of which embryos implanted and were delivered after multiple ET were 100% consistent with previously validated embryo DNA fingerprinting-based assignments. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

    PubMed Central

    Bibikova, Marina; Wickham-Garcia, Eliza; Agirre, Xabier; Alvarez, Sara; Brüggemann, Monika; Bug, Stefanie; Calasanz, Maria J.; Deckert, Martina; Dreyling, Martin; Du, Ming Q.; Dürig, Jan; Dyer, Martin J. S.; Fan, Jian-Bing; Gesk, Stefan; Hansmann, Martin-Leo; Harder, Lana; Hartmann, Sylvia; Klapper, Wolfram; Küppers, Ralf; Montesinos-Rongen, Manuel; Nagel, Inga; Pott, Christiane; Richter, Julia; Román-Gómez, José; Seifert, Marc; Stein, Harald; Suela, Javier; Trümper, Lorenz; Vater, Inga; Prosper, Felipe; Haferlach, Claudia; Cigudosa, Juan Cruz; Siebert, Reiner

    2009-01-01

    Background Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play

  14. A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

    PubMed

    Ise, Ryota; Uehara, Shotaro; Akiyama, Hideo; Kondo, Satoshi; Iwasaki, Kazuhide; Nagata, Ryoichi; Nobumasa, Hitoshi; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-06-01

    Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

  15. In Silico Analysis of Microarray-Based Gene Expression Profiles Predicts Tumor Cell Response to Withanolides

    PubMed Central

    Efferth, Thomas; Greten, Henry Johannes

    2012-01-01

    Withania somnifera (L.) Dunal (Indian ginseng, winter cherry, Solanaceae) is widely used in traditional medicine. Roots are either chewed or used to prepare beverages (aqueous decocts). The major secondary metabolites of Withania somnifera are the withanolides, which are C-28-steroidal lactone triterpenoids. Withania somnifera extracts exert chemopreventive and anticancer activities in vitro and in vivo. The aims of the present in silico study were, firstly, to investigate whether tumor cells develop cross-resistance between standard anticancer drugs and withanolides and, secondly, to elucidate the molecular determinants of sensitivity and resistance of tumor cells towards withanolides. Using IC50 concentrations of eight different withanolides (withaferin A, withaferin A diacetate, 3-azerininylwithaferin A, withafastuosin D diacetate, 4-B-hydroxy-withanolide E, isowithanololide E, withafastuosin E, and withaperuvin) and 19 established anticancer drugs, we analyzed the cross-resistance profile of 60 tumor cell lines. The cell lines revealed cross-resistance between the eight withanolides. Consistent cross-resistance between withanolides and nitrosoureas (carmustin, lomustin, and semimustin) was also observed. Then, we performed transcriptomic microarray-based COMPARE and hierarchical cluster analyses of mRNA expression to identify mRNA expression profiles predicting sensitivity or resistance towards withanolides. Genes from diverse functional groups were significantly associated with response of tumor cells to withaferin A diacetate, e.g. genes functioning in DNA damage and repair, stress response, cell growth regulation, extracellular matrix components, cell adhesion and cell migration, constituents of the ribosome, cytoskeletal organization and regulation, signal transduction, transcription factors, and others. PMID:27605335

  16. An event-specific DNA microarray to identify genetically modified organisms in processed foods.

    PubMed

    Kim, Jae-Hwan; Kim, Su-Youn; Lee, Hyungjae; Kim, Young-Rok; Kim, Hae-Yeong

    2010-05-26

    We developed an event-specific DNA microarray system to identify 19 genetically modified organisms (GMOs), including two GM soybeans (GTS-40-3-2 and A2704-12), thirteen GM maizes (Bt176, Bt11, MON810, MON863, NK603, GA21, T25, TC1507, Bt10, DAS59122-7, TC6275, MIR604, and LY038), three GM canolas (GT73, MS8xRF3, and T45), and one GM cotton (LLcotton25). The microarray included 27 oligonucleotide probes optimized to identify endogenous reference targets, event-specific targets, screening targets (35S promoter and nos terminator), and an internal target (18S rRNA gene). Thirty-seven maize-containing food products purchased from South Korean and US markets were tested for the presence of GM maize using this microarray system. Thirteen GM maize events were simultaneously detected using multiplex PCR coupled with microarray on a single chip, at a limit of detection of approximately 0.5%. Using the system described here, we detected GM maize in 11 of the 37 food samples tested. These results suggest that an event-specific DNA microarray system can reliably detect GMOs in processed foods.

  17. A multichannel order-statistic technique for cDNA microarray image processing.

    PubMed

    Lukac, Rastislav; Plataniotis, Konstantinos N; Smolka, Bogdan; Venetsanopoulos, Anastasios N

    2004-12-01

    This paper introduces an automated image processing procedure capable of processing complementary deoxyribonucleic acid (cDNA) microarray images. Microarray data is contaminated by noise and suffers from broken edges and visual artifacts. Without the utilization of a filter, subsequent tasks such as spot identification and gene expression determination cannot be completed. By employing, in a unique cascade processing cycle, nonlinear filtering solutions based on robust order statistics, the procedure: 1) removes both background and high-frequency corrupting noise and 2) correctly identifies edges and spots in cDNA microarray data. The proposed solution operates directly on the microarray data, does not rely on explicit data normalization or spot separation preprocessing, and operates in a robust manner without using heuristically determined design parameters. Other routine microarray processing operations such as shape manipulations and grid adjustments can be used in conjunction with the developed solution in the processing pipeline. Experimentation reported in this paper indicates that the proposed solution yields excellent performance by removing noise and enhancing spot location determination.

  18. Toward 'smart' DNA microarrays: algorithms for improving data quality and statistical inference

    NASA Astrophysics Data System (ADS)

    Bakewell, David J. G.; Wit, Ernst

    2007-12-01

    DNA microarrays are a laboratory tool for understanding biological processes at the molecular scale and future applications of this technology include healthcare, agriculture, and environment. Despite their usefulness, however, the information microarrays make available to the end-user is not used optimally, and the data is often noisy and of variable quality. This paper describes the use of hierarchical Maximum Likelihood Estimation (MLE) for generating algorithms that improve the quality of microarray data and enhance statistical inference about gene behavior. The paper describes examples of recent work that improves microarray performance, demonstrated using data from both Monte Carlo simulations and published experiments. One example looks at the variable quality of cDNA spots on a typical microarray surface. It is shown how algorithms, derived using MLE, are used to "weight" these spots according to their morphological quality, and subsequently lead to improved detection of gene activity. Another example, briefly discussed, addresses the "noisy data about too many genes" issue confronting many analysts who are also interested in the collective action of a group of genes, often organized as a pathway or complex. Preliminary work is described where MLE is used to "share" variance information across a pre-assigned group of genes of interest, leading to improved detection of gene activity.

  19. Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates▿

    PubMed Central

    Zhu, Ling-Xiang; Zhang, Zhi-Wei; Wang, Can; Yang, Hua-Wei; Jiang, Di; Zhang, Qiong; Mitchelson, Keith; Cheng, Jing

    2007-01-01

    We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLSB) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of the mecA, blaZ (methicillin and penicillin resistance), aac(6′)-Ie-aph(2‴) (aminoglycoside resistance), ermA and ermC genes (MLSB resistance), and the msrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate between Staphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically important S. aureus and 237 CoNS isolates, with correlations of 100% for S. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17 mecA-positive CoNS and 60 aac(6′)-Ie-aph(2‴)-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (≤5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies. PMID:17728472

  20. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

    PubMed

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

    2004-12-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

  1. DNA Microarray-Based Screening and Characterization of Traditional Chinese Medicine

    PubMed Central

    Kiyama, Ryoiti

    2017-01-01

    The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterization of traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway-based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields. PMID:28146102

  2. Quantitative comparison of the HSV-1 and HSV-2 transcriptomes using DNA microarray analysis

    SciTech Connect

    Aguilar, J.S. . E-mail: jsaguila@uci.edu; Devi-Rao, G.V.; Rice, M.K.; Sunabe, J.; Ghazal, P.; Wagner, E.K.

    2006-04-25

    The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U{sub L}4, U{sub L}29, U{sub L}30, and U{sub L}31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.

  3. Modeling the temporal evolution of the Drosophila gene expression from DNA microarray time series

    NASA Astrophysics Data System (ADS)

    Haye, Alexandre; Dehouck, Yves; Kwasigroch, Jean Marc; Bogaerts, Philippe; Rooman, Marianne

    2009-03-01

    The time evolution of gene expression across the developmental stages of the host organism can be inferred from appropriate DNA microarray time series. Modeling this evolution aims eventually at improving the understanding and prediction of the complex phenomena that are the basis of life. We focus on the embryonic-to-adult development phases of Drosophila melanogaster, and chose to model the expression network with the help of a system of differential equations with constant coefficients, which are nonlinear in the transcript concentrations but linear in their logarithms. To reduce the dimensionality of the problem, genes having similar expression profiles are grouped into 17 clusters. We show that a simple linear model is able to reproduce the experimental data with very good precision, owing to the large number of parameters that represent the connections between the clusters. Remarkably, the parameter reduction allowed elimination of up to 80-85% of these connections while keeping fairly good precision. This result supports the low-connectivity hypothesis of gene expression networks, with about three connections per cluster, without introducing a priori hypotheses. The core of the network shows a few gene clusters with negative self-regulation, and some highly connected clusters involving proteins with crucial functions.

  4. Optimization of high-density cDNA-microarray protocols by ‘design of experiments’

    PubMed Central

    Wrobel, Gunnar; Schlingemann, Joerg; Hummerich, Lars; Kramer, Heidi; Lichter, Peter; Hahn, Meinhard

    2003-01-01

    Expression analysis using microarray technology implies a complex experimental procedure with a large number of parameters affecting the final result. We have demonstrated that optimization of such a complex protocol can be far better handled using design of experiments (DOE) than by working on a single parameter at a time. Based on the results of a screening design, we developed a spotting buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 2-fold increase in signal-to-background ratio compared to 3× SSC. Comparison to seven other buffers tested on 10 different substrates revealed it had the highest sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated microscope slides at a density of up to 70 000 spots per slide. A second DOE approach characterized the RNA labeling process with regard to the concentration of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, produced an improvement in the performance of the labeling procedure by a factor of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of establishing a stable expression profiling protocol and its further optimization can be significantly shortened and improved by DOE. PMID:12799456

  5. A gDNA microarray for genotyping salvia species.

    PubMed

    Olarte, Alexandra; Mantri, Nitin; Nugent, Gregory; Wohlmuth, Hans; Li, Chun Guang; Xue, Charlie; Pang, Edwin

    2013-07-01

    Salvia is an important genus from the Lamiaceae with approximately 1,000 species. This genus is distributed globally and cultivated for ornamental, culinary, and medicinal uses. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia subtracted diversity array (SDA) a suppression subtractive hybridization (SSH) was performed between a pool of Salvia species and a pool of angiosperms and non-angiosperms to selectively isolate Salvia-specific sequences. A total of 285-subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating S. officinalis and S. miltiorrhiza from their closely related species and was also able to reveal genetic relationships consistent with geographical origins. In addition, this approach was capable of isolating highly polymorphic sequences from chloroplast and nuclear DNA without preliminary sequence information. Therefore, SDA is a powerful technique for fingerprinting non-model plants and for identifying new polymorphic loci that may be developed as potential molecular markers.

  6. Phenotype Microarray Profiling of Zymomonas mobilis ZM4

    SciTech Connect

    Bochner, Barry; Gomez, Vanessa; Ziman, michael; Yang, Shihui; Brown, Steven D

    2009-01-01

    In this study, we developed a Phenotype MicroArray{trademark} (PM) protocol to profile cellular phenotypes in Zymomonas mobilis, which included a standard set of nearly 2,000 assays for carbon, nitrogen, phosphorus and sulfur source utilization, nutrient stimulation, pH and osmotic stresses, and chemical sensitivities with 240 inhibitory chemicals. We observed two positive assays for C-source utilization (fructose and glucose) using the PM screen, which uses redox chemistry and cell respiration as a universal reporter to profile growth phenotypes in a high-throughput 96-well plate-based format. For nitrogen metabolism, the bacterium showed a positive test results for ammonia, aspartate, asparagine, glutamate, glutamine, and peptides. Z. mobilis appeared to use a diverse array of P-sources with two exceptions being pyrophosphate and tripolyphosphate. The assays suggested that Z. mobilis uses both inorganic and organic compounds as S-sources. No stimulation by nutrients was detected; however, there was evidence of partial inhibition by purines and pyrimidines, NAD, and deferoxamine. Z. mobilis was relatively resistant to acid pH, tolerating a pH down to about 4.0. It also tolerated phosphate, sulfate, and nitrate, but was rather sensitive to chloride and nitrite. Z. mobilis showed resistance to a large number of diverse chemicals that inhibit most bacteria. The information from PM analysis provides an overview of Z. mobilis physiology and a foundation for future comparisons of other wild-type and mutant Z. mobilis strains.

  7. Candidate genes involved in tanshinone biosynthesis in hairy roots of Salvia miltiorrhiza revealed by cDNA microarray.

    PubMed

    Cui, Guanghong; Huang, Luqi; Tang, Xiaojing; Zhao, Jingxue

    2011-04-01

    Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.

  8. Selective immobilization and detection of DNA on biopolymer supports for the design of microarrays.

    PubMed

    Kargl, R; Vorraber, V; Ribitsch, V; Köstler, S; Stana-Kleinschek, K; Mohan, T

    2015-06-15

    DNA immobilization for the manufacturing of microarrays requires sufficient probe density, low unspecific binding and high interaction efficiency with complementary strands that are detected from solutions. Many of these important parameters are affected by the surface chemistry and the blocking steps conducted during DNA spotting and hybridization. This work describes an alternative method to selectively immobilize probes and to detect DNA on biocompatible, hydrophilic cellulose coated supports with low unspecific binding, high selectivity and appropriate sensitivity. It takes advantage of a relatively selective adsorption of water soluble polysaccharides on a solid cellulose matrix. Single strands of DNA were conjugated to this soluble polysaccharide and subsequently micro-spotted on solid cellulose thin films that were coated on glass and polymer slides. This resulted in adsorptively bound DNA-probes that were used to detect complementary, labelled DNA strands with different lengths and sequences by hybridization. The interaction of the DNA-conjugates with cellulose surfaces and the selectivity of hybridization were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence scanning. The method of non-covalent immobilization of DNA probes on an uncharged, non-reactive, hydrophilic support lowers the unspecific binding and the number of handling steps required to conduct the experiments for the detection of DNA on microarrays. Simultaneously selectivity, hybridization efficiency and detection limits are maintained. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Xylella fastidiosa gene expression analysis by DNA microarrays

    PubMed Central

    2009-01-01

    Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants. PMID:21637690

  10. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    PubMed

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  11. DNA methylation profiling can classify HIV-associated lymphomas.

    PubMed

    Matsunaga, Akihiro; Hishima, Tsunekazu; Tanaka, Noriko; Yamasaki, Maria; Yoshida, Lui; Mochizuki, Makoto; Tanuma, Junko; Oka, Shinichi; Ishizaka, Yukihito; Shimura, Mari; Hagiwara, Shotaro

    2014-02-20

    HIV-positive patients have a 60-fold to 200-fold increased incidence of non-Hodgkin lymphomas, including Burkitt lymphoma, diffuse large B-cell lymphoma, and primary central nervous system lymphoma. HIV-associated lymphomas frequently have features such as extranodal involvement, decreased responses to standard chemotherapy, and high relapse rates, which indicate a poor prognosis. General pathological features do not clearly differentiate HIV-associated lymphomas from non-HIV lymphomas. To investigate the features of HIV-associated lymphomas, we performed genome-wide DNA methylation profiling of HIV and non-HIV lymphomas using Illumina GoldenGate Methylation Cancer Panel I and Illumina Infinium HumanMethylation450 BeadChip microarrays. DNA methylation profiles in HIV-associated and non-HIV lymphomas were characterized using unsupervised hierarchical clustering analyses. The analyses of promoter regions revealed unique DNA methylation profiles in HIV-associated lymphomas, suggesting profile differences compared with non-HIV lymphomas, which implies specific gene regulation in HIV-associated lymphoma involving DNA methylation. Based on HumanMethylation450 BeadChip data, 2541 target sites were selected as differing significantly in comparisons between HIV-associated and non-HIV-associated lymphomas using Wilcoxon's rank-sum test (P <0.05) and Δβ values more than 0.30. Recurrent cases of HIV-associated lymphoma had different profiles compared with nonrecurrent HIV lymphomas. DNA methylation profiling indicated that 2541 target sites differed significantly in HIV-associated lymphoma, which may partly explain the poor prognosis. Our data indicate that the methylation profiles of target genes have potential in elucidating HIV-associated lymphomagenesis and can serve as new prognostic markers.

  12. Recognition of multiple imbalanced cancer types based on DNA microarray data using ensemble classifiers.

    PubMed

    Yu, Hualong; Hong, Shufang; Yang, Xibei; Ni, Jun; Dan, Yuanyuan; Qin, Bin

    2013-01-01

    DNA microarray technology can measure the activities of tens of thousands of genes simultaneously, which provides an efficient way to diagnose cancer at the molecular level. Although this strategy has attracted significant research attention, most studies neglect an important problem, namely, that most DNA microarray datasets are skewed, which causes traditional learning algorithms to produce inaccurate results. Some studies have considered this problem, yet they merely focus on binary-class problem. In this paper, we dealt with multiclass imbalanced classification problem, as encountered in cancer DNA microarray, by using ensemble learning. We utilized one-against-all coding strategy to transform multiclass to multiple binary classes, each of them carrying out feature subspace, which is an evolving version of random subspace that generates multiple diverse training subsets. Next, we introduced one of two different correction technologies, namely, decision threshold adjustment or random undersampling, into each training subset to alleviate the damage of class imbalance. Specifically, support vector machine was used as base classifier, and a novel voting rule called counter voting was presented for making a final decision. Experimental results on eight skewed multiclass cancer microarray datasets indicate that unlike many traditional classification approaches, our methods are insensitive to class imbalance.

  13. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays.

    PubMed

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  14. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays

    PubMed Central

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  15. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    PubMed

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  16. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

    PubMed

    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

  17. DNA Microarrays for Aptamer Identification and Structural Characterization

    DTIC Science & Technology

    2012-09-01

    Zhou, T., Gulari, E., “ Microfluidic Reactor Array Device for Massively Parallel in Situ Synthesis of Oligonucleotides,” Sensors and Actuators B...Soh, H. T., “Quantitative Selection of DNA Aptamers Through Microfluidic Selection and High- throughput Sequencing,” Proc Natl Acad Sci., 107, 2010

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

    PubMed

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

    2016-07-01

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

  19. cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)

    PubMed Central

    Quinn, Patrick; Bowers, Robert M.; Zhang, Xiaoyu; Wahlund, Thomas M.; Fanelli, Michael A.; Olszova, Daniela; Read, Betsy A.

    2006-01-01

    Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis. PMID:16885305

  20. A three-dimensional waveguide substrate for DNA-microarrays based on macroporous silicon

    NASA Astrophysics Data System (ADS)

    Dertinger, Stephan K.; Klühr, Marco; Sauermann, Alexander; Thein, Kerstin

    2005-06-01

    In this paper we present a three-dimensional waveguide structure with unique optical and fluidic properties and demonstrate its application as a substrate for DNA microarrays. The structure is fabricated by thermal oxidation of a macroporous silicon membrane with a periodic pattern of discrete microchannels running perpendicular through the substrate. Partial oxidation generates compartments with channel walls that are completely converted into SiO2 but leaves a rectangular grid of silicon walls separating the SiO2 compartments. We demonstrate that the SiO2 walls act as optical waveguides and the opaque silicon walls divide the substrate into optically isolated compartments. In DNA microarray experiments, we show that the silicon walls of the compartments prevent cross talk between adjacent DNA spots. The structure is compatible with all conventional read-out techniques such as fluorescence, chemiluminescence, and precipitation staining.

  1. Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

    PubMed

    Sharma, Himanshu; Wood, Jennifer B; Lin, Sophia; Corn, Robert M; Khine, Michelle

    2014-09-23

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

  2. Controlling microarray DNA hybridization efficiency by probe-surface distance and external surface electrostatics

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

    DNA microarrays are analytical devices designed to determine the composition of multicomponent solutions of nucleic acids, DNA or RNA. These devices are promising technology for diverse applications, including sensing, diagnostics, and drug/gene delivery. Here, we modify a hybridization adsorption isotherm to study the effects of probe-surface distance and the external electrostatic fields, on the oligonucleotide hybridization in microarray and how these effects are varies depending on surface probe density and target concentration. This study helps in our understanding on-surface hybridization mechanisms, and from it we can observe a significant effect of the probe-surface distance, and the external electrostatic fields, on the hybridization yield. In addition we present a simple new criteria to control the oligonucleotide hybridization efficiency by providing a chart illustrating the effects of all factors on the DNA-hybridization efficiency.

  3. Long Noncoding RNA Expression Profiling Using Arraystar LncRNA Microarrays.

    PubMed

    Shi, Yanggu; Shang, Jindong

    2016-01-01

    Arraystar LncRNA microarrays are designed for global gene expression profiling of both LncRNAs and mRNAs on the same array. The array contents feature comprehensive collections of LncRNAs and include entire sets of known coding mRNAs. Each RNA transcript is detected by a splice junction-specific probe or a unique exon sequence, such that the alternatively spliced transcript isoforms or variants are reliably and accurately detected. The highly optimized experimental protocols and efficient workflow ensure sensitive, robust, and accurate microarray data generation. Standard data analyses are provided for microarray raw data processing, data quality control, gene expression clustering and heat map visualization, differentially expressed LncRNAs and mRNAs, LncRNA subcategories, regulatory relationships of LncRNAs with the mRNAs, gene ontology, and pathway analysis. The LncRNA microarrays are powerful tools for the study of LncRNAs in biology and disease, with broad applications in gene expression profiling, gene regulatory mechanism research, LncRNA functional discovery, and biomarker development.

  4. Microarrays in the 2010s: the contribution of microarray-based gene expression profiling to breast cancer classification, prognostication and prediction

    PubMed Central

    2011-01-01

    Breast cancer comprises a collection of diseases with distinctive clinical, histopathological, and molecular features. Importantly, tumors with similar histological features may display disparate clinical behaviors. Gene expression profiling using microarray technologies has improved our understanding of breast cancer biology and has led to the development of a breast cancer molecular taxonomy and of multigene 'signatures' to predict outcome and response to systemic therapies. The use of these prognostic and predictive signatures in routine clinical decision-making remains controversial. Here, we review the clinical relevance of microarray-based profiling of breast cancer and discuss its impact on patient management. PMID:21787441

  5. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates

    PubMed Central

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

    2014-01-01

    Streptococcus agalactiae frequently colonizes the urogenital tract, and it is a major cause of bacterial septicemia, meningitis, and pneumonia in newborns. For typing purposes, a microarray targeting group B streptococcus (GBS) virulence-associated markers and resistance genes was designed and validated with reference strains, as well as clinical and veterinary isolates. Selected isolates were also subjected to multilocus sequence typing. It was observed that putative typing markers, such as alleles of the alpha-like protein or capsule types, vary independently of each other, and they also vary independently from the affiliation to their multilocus sequence typing (MLST)-defined sequence types. Thus, it is not possible to assign isolates to sequence types based on the identification of a single distinct marker, such as a capsule type or alp allele. This suggests the occurrence of frequent genomic recombination. For array-based typing, a set of 11 markers (bac, alp, pil1 locus, pepS8, fbsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2, and rgfC/A/D/B) was defined that provides a framework for splitting the tested 448 S. agalactiae isolates into 76 strains that clustered mainly according to MLST-defined clonal complexes. There was evidence for region- and host-specific differences in the population structure of S. agalactiae, as well as an overrepresentation of strains related to sequence type 17 among the invasive isolates. The arrays and typing scheme described here proved to be a convenient tool for genotyping large numbers of clinical/veterinary isolates and thus might help obtain insight into the epidemiology of S. agalactiae. PMID:25165085

  6. ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

    PubMed Central

    Moulton, Kevin R.; Taylor, Amber W.; Rowlen, Kathy L.; Dawson, Erica D.

    2011-01-01

    DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective. The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is ˜1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection. Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one

  7. A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

    PubMed Central

    2010-01-01

    Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification. PMID:21062433

  8. Forensic DNA Profiling and Database

    PubMed Central

    Panneerchelvam, S.; Norazmi, M.N.

    2003-01-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

  9. The role of DNA microarrays in the evaluation of fetal death.

    PubMed

    Reddy, Uma M; Page, Grier P; Saade, George R

    2012-04-01

    Fetal death occurs in 15% of clinically recognized pregnancies. Cytogenetic abnormalities are present in 50% of spontaneous abortions (fetal deaths < 20 weeks) whereas the rate is 6% to 13% for stillbirths (fetal deaths ≥ 20 weeks). Microarray has been demonstrated to increase the diagnosis of genetic abnormalities by providing coverage of the entire genome at a higher density, detecting as small as 50 to 100 kb deletions or duplications, known as copy number changes. Microarray is particularly suited for evaluation of fetal death because DNA can still be analyzed in macerated fetuses and nonviable tissue, two situations where culturing and karyotyping is known to have low yield. Microarray has already proven successful in providing additional genetic information beyond karyotype in spontaneous abortion. The few studies on the use of microarray in stillbirth evaluation have been promising, demonstrating an increase in the diagnosis of clinically relevant genetic abnormalities when compared with karyotype. As the cost and technology improve, microarray may ultimately become the first line screen for genetic abnormalities in stillbirth. The accurate diagnosis of a genetic abnormality as the cause for fetal death may provide closure for families, prevent unnecessary treatments, and enable clinicians to more accurately counsel and manage subsequent pregnancies.

  10. Recent Applications of DNA Microarray Technology to Toxicology and Ecotoxicology

    PubMed Central

    Lettieri, Teresa

    2006-01-01

    Gene expression is a unique way of characterizing how cells and organisms adapt to changes in the external environment. The measurements of gene expression levels upon exposure to a chemical can be used both to provide information about the mechanism of action of the toxicant and to form a sort of “genetic signature” for the identification of toxic products. The development of high-quality, commercially available gene arrays has allowed this technology to become a standard tool in molecular toxicology. Several national and international initiatives have provided the proof-of-principle tests for the application of gene expression for the study of the toxicity of new and existing chemical compounds. In the last few years the field has progressed from evaluating the potential of the technology to illustrating the practical use of gene expression profiling in toxicology. The application of gene expression profiling to ecotoxicology is at an earlier stage, mainly because of the the many variables involved in analyzing the status of natural populations. Nevertheless, significant studies have been carried out on the response to environmental stressors both in model and in nonmodel organisms. It can be easily predicted that the development of stressor-specific signatures in gene expression profiling in ecotoxicology will have a major impact on the ecotoxicology field in the near future. International collaborations could play an important role in accelerating the application of genomic approaches in ecotoxicology. PMID:16393650

  11. SNP-microarrays can accurately identify the presence of an individual in complex forensic DNA mixtures.

    PubMed

    Voskoboinik, Lev; Ayers, Sheri B; LeFebvre, Aaron K; Darvasi, Ariel

    2015-05-01

    Common forensic and mass disaster scenarios present DNA evidence that comprises a mixture of several contributors. Identifying the presence of an individual in such mixtures has proven difficult. In the current study, we evaluate the practical usefulness of currently available "off-the-shelf" SNP microarrays for such purposes. We found that a set of 3000 SNPs specifically selected for this purpose can accurately identify the presence of an individual in complex DNA mixtures of various compositions. For example, individuals contributing as little as 5% to a complex DNA mixture can be robustly identified even if the starting DNA amount was as little as 5.0ng and had undergone whole-genome amplification (WGA) prior to SNP analysis. The work presented in this study represents proof-of-principle that our previously proposed approach, can work with real "forensic-type" samples. Furthermore, in the absence of a low-density focused forensic SNP microarray, the use of standard, currently available high-density SNP microarrays can be similarly used and even increase statistical power due to the larger amount of available information.

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

    PubMed Central

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

    2004-01-01

    Background Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. Results We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. Conclusions We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase. PMID:15610561

  13. Use of a DNA microarray for detection and identification of bacterial pathogens associated with fishery products.

    PubMed

    Cao, Boyang; Li, Rongrong; Xiong, Songjin; Yao, Fangfang; Liu, Xiangqian; Wang, Min; Feng, Lu; Wang, Lei

    2011-12-01

    We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

  14. Electronic microarray analysis of 16S rDNA amplicons for bacterial detection.

    PubMed

    Barlaan, Edward A; Sugimori, Miho; Furukawa, Seiji; Takeuchi, Kazuhisa

    2005-01-12

    Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.

  15. A novel neural network approach to cDNA microarray image segmentation.

    PubMed

    Wang, Zidong; Zineddin, Bachar; Liang, Jinling; Zeng, Nianyin; Li, Yurong; Du, Min; Cao, Jie; Liu, Xiaohui

    2013-07-01

    Microarray technology has become a great source of information for biologists to understand the workings of DNA which is one of the most complex codes in nature. Microarray images typically contain several thousands of small spots, each of which represents a different gene in the experiment. One of the key steps in extracting information from a microarray image is the segmentation whose aim is to identify which pixels within an image represent which gene. This task is greatly complicated by noise within the image and a wide degree of variation in the values of the pixels belonging to a typical spot. In the past there have been many methods proposed for the segmentation of microarray image. In this paper, a new method utilizing a series of artificial neural networks, which are based on multi-layer perceptron (MLP) and Kohonen networks, is proposed. The proposed method is applied to a set of real-world cDNA images. Quantitative comparisons between the proposed method and commercial software GenePix(®) are carried out in terms of the peak signal-to-noise ratio (PSNR). This method is shown to not only deliver results comparable and even superior to existing techniques but also have a faster run time.

  16. Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling

    SciTech Connect

    R. Quatrini; C. Appia-Ayme; Y. Denis; J. Ratouchniak; F. Veloso; J. Valdes; C. Lefimil; S. Silver; F. Roberto; O. Orellana; F. Denizot; E. Jedlicki; D. Holmes; V. Bonnefoy

    2006-09-01

    Acidithiobacillus ferrooxidans is a well known acidophilic, chemolithoautotrophic, Gram negative, bacterium involved in bioleaching and acid mine drainage. In aerobic conditions, it gains energy mainly from the oxidation of ferrous iron and/or reduced sulfur compounds present in ores. After initial oxidation of the substrate, electrons from ferrous iron or sulfur enter respiratory chains and are transported through several redox proteins to oxygen. However, the oxidation of ferrous iron and reduced sulfur compounds has also to provide electrons for the reduction of NAD(P) that is subsequently required for many metabolic processes including CO2 fixation. To help to unravel the enzymatic pathways and the electron transfer chains involved in these processes, a genome-wide microarray transcript profiling analysis was carried out. Oligonucleotides corresponding to approximately 3000 genes of the A. ferrooxidans type strain ATCC23270 were spotted onto glass-slides and hybridized with cDNA retrotranscribed from RNA extracted from ferrous iron and sulfur grown cells. The genes which are preferentially transcribed in ferrous iron conditions and those preferentially transcribed in sulfur conditions were analyzed. The expression of a substantial number of these genes has been validated by real-time PCR, Northern blot hybridization and/or immunodetection analysis. Our results support and extend certain models of iron and sulfur oxidation and highlight previous observations regarding the possible presence of alternate electron pathways. Our findings also suggest ways in which iron and sulfur oxidation may be co-ordinately regulated. An accompanying paper (Appia-Ayme et al.) describes results pertaining to other metabolic functions.

  17. An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

    PubMed Central

    Price, Jordan V.; Tabakman, Scott M.; Li, Yanguang; Gong, Ming; Hong, Guosong; Feng, Ju; Utz, Paul J.; Dai, Hongjie

    2013-01-01

    High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone. PMID:23923050

  18. Role of DNA profiling in forensic odontology

    PubMed Central

    Sakari, S. Leena; Jimson, Sudha; Masthan, K. M. K.; Jacobina, Jenita

    2015-01-01

    The recent advances in DNA profiling have made DNA evidence to be more widely accepted in courts. This has revolutionized the aspect of forensic odontology. DNA profiling/DNA fingerprinting has come a long way from the conventional fingerprints. DNA that is responsible for all the cell's activities, yields valuable information both in the healthy and diseased individuals. When other means of traditional identification become impossible following mass calamities or fire explosions, teeth provide a rich source of DNA as they have a high chemical as well as physical resistance. The recent evolution in the isolation of DNA and the ways of running a DNA fingerprint are highlighted in this literature review. PMID:26015692

  19. Conventional and nanotechniques for DNA methylation profiling.

    PubMed

    Shanmuganathan, Rajasree; Basheer, Nazeema B; Amirthalingam, Laxmi; Muthukumar, Harshiny; Kaliaperumal, Rajendran; Shanmugam, Kumaran

    2013-01-01

    DNA methylation is critical for gene silencing and is associated with the incidence of many diseases, including cancer. Underlying molecular mechanisms of human diseases and tissue-specific gene expression have been elucidated based on DNA methylation studies. This review highlights the advantages and drawbacks of various methylation screening techniques: blotting, genomic sequencing, bisulfite sequencing, methylation-specific PCR, methylated DNA immunoprecipitation, microarray analysis, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, nanowire transistor detection procedure, quantum dot-based nanoassay, single-molecule real-time detection, fluorimetric assay, electrochemical detection, and atomic force spectroscopy. The review provides insight for selecting a method or a combination of methods for DNA methylation analysis. Convergence of conventional and contemporary nanotechniques to enumerate methylation at specific CpG sites of oncogene would fill the gap in diagnosis of cancer.

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

    PubMed

    Bertagnolli, Nicolas M; Drake, Justin A; Tennessen, Jason M; Alter, Orly

    2013-01-01

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

  1. Understanding the molecular aspects of oriental obesity pattern differentiation using DNA microarray.

    PubMed

    Hong, Sun Woo; Yoo, Jae-Wook; Bose, Shambhunath; Park, Jung-Hyun; Han, Kyungsun; Kim, Soyoun; Lim, Chi-Yeon; Kim, Hojun; Lee, Dong-Ki

    2015-10-19

    Human constitution, the fundamental basis of oriental medicine, is categorized into different patterns for a particular disease according to the physical, physiological, and clinical characteristics of the individuals. Obesity, a condition of metabolic disorder, is classified according to six patterns in oriental medicine, as follows: spleen deficiency syndrome, phlegm fluid syndrome, yang deficiency syndrome (YDS), food accumulation syndrome (FAS), liver depression syndrome (LDS), and blood stasis syndrome. In oriental medicine, identification of the disease pattern for individual obese patients is performed on the basis of differentiation in obesity syndrome index and, accordingly, personalized treatment is provided to the patients. The aim of the current study was to understand the obesity patterns in oriental medicine from the genomic point of view via determining the gene expression signature of obese patients using peripheral blood mononuclear cells as the samples. The study was conducted in 23 South Korean obese subjects (19 female and four male) with BMI ≥25 kg/m(2). Identification of oriental obesity pattern was based on the software-guided evaluation of the responses of the subjects to a questionnaire developed by the Korean Institute of Oriental Medicine. The expression profiles of genes were determined using DNA microarray and the level of transcription of genes of interest was further evaluated using quantitative real-time PCR (qRT-PCR). Gene clustering analysis of the microarray data from the FAS, LDS, and YDS subjects exhibited disease pattern-specific upregulation of expression of several genes in a particular cluster. Further analysis of transcription of selected genes using qRT-PCR led to identification of specific genes, including prostaglandin endoperoxide synthase 2, G0/G1 switch 2, carcinoembryonic antigen-related cell adhesion molecule 3, cystein-serine-rich nuclear protein 1, and interleukin 8 receptor, alpha which were highly expressed in

  2. An MCMC Algorithm for Target Estimation in Real-Time DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Vikalo, Haris; Gokdemir, Mahsuni

    2010-12-01

    DNA microarrays detect the presence and quantify the amounts of nucleic acid molecules of interest. They rely on a chemical attraction between the target molecules and their Watson-Crick complements, which serve as biological sensing elements (probes). The attraction between these biomolecules leads to binding, in which probes capture target analytes. Recently developed real-time DNA microarrays are capable of observing kinetics of the binding process. They collect noisy measurements of the amount of captured molecules at discrete points in time. Molecular binding is a random process which, in this paper, is modeled by a stochastic differential equation. The target analyte quantification is posed as a parameter estimation problem, and solved using a Markov Chain Monte Carlo technique. In simulation studies where we test the robustness with respect to the measurement noise, the proposed technique significantly outperforms previously proposed methods. Moreover, the proposed approach is tested and verified on experimental data.

  3. VARAN: a web server for variability analysis of DNA microarray experiments.

    PubMed

    Golfier, G; Dang, M Tran; Dauphinot, L; Graison, E; Rossier, J; Potier, M-C

    2004-07-10

    Here, we describe a tool for VARiability Analysis of DNA microarrays experiments (VARAN), a freely available Web server that performs a signal intensity based analysis of the log2 expression ratio variability deduced from DNA microarray data (one or two channels). Two modules are proposed: VARAN generator to compute a sliding windows analysis of the experimental variability (mean and SD) and VARAN analyzer to compare experimental data with an asymptotic variability model previously built with the generator module from control experiments. Both modules provide normalized intensity signals with five possible methods, log ratio values and a list of genes showing significant variations between conditions. http://www.bionet.espci.fr/varan/ http://www.bionet.espci.fr/varan/help.html

  4. DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae.

    PubMed

    Yasokawa, Daisuke; Murata, Satomi; Iwahashi, Yumiko; Kitagawa, Emiko; Kishi, Katsuyuki; Okumura, Yukihiro; Iwahashi, Hitoshi

    2010-05-01

    Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation.

  5. Assessing Differential Expression Measurements by Highly Parallel Pyrosequencing and DNA Microarrays: A Comparative Study

    PubMed Central

    Ariño, Joaquín; Casamayor, Antonio; Pérez, Julián Perez; Pedrola, Laia; Álvarez-Tejado, Miguel; Marbà, Martina; Santoyo, Javier

    2013-01-01

    Abstract To explore the feasibility of pyrosequencing for quantitative differential gene expression analysis we have performed a comparative study of the results of the sequencing experiments to those obtained by a conventional DNA microarray platform. A conclusion from our analysis is that, over a threshold of 35 normalized reads per gene, the measurements of gene expression display a good correlation with the references. The observed concordance between pyrosequencing and DNA microarray platforms beyond the threshold was of 0.8, measured as a Pearson's correlation coefficient. In differential gene expression the initial aim is the quantification the differences among transcripts when comparing experimental conditions. Thus, even in a scenario of low coverage the concordance in the measurements is quite acceptable. On the other hand, the comparatively longer read size obtained by pyrosequencing allows detecting unconventional splicing forms. PMID:21919703

  6. DNA Microarray and Proteomic Strategies for Understanding Alcohol Action

    PubMed Central

    Sikela, James M.; MacLaren, Erik J.; Kim, Young; Karimpour-Fard, Anis; Cai, Wei-Wen; Pollack, Jonathan; Hitzemann, Robert; Belknap, John; McWeeney, Shannon; Kerns, Robnet T.; Downing, Chris; Johnson, Thomas E.; Grant, Kathleen J.; Tabakoff, Boris; Hoffman, Paula; Wu, Christine C.; Miles, Michael F.

    2009-01-01

    This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant. PMID:16573589

  7. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    PubMed

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

    PubMed Central

    Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  9. DNA microarray analysis of the liver of mice treated with cobalt chloride.

    PubMed

    Matsumoto, Kanako; Fujishiro, Hitomi; Satoh, Masahiko; Himeno, Seiichiro

    2010-12-01

    To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regulated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon.

  10. Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

    PubMed Central

    Mahalingam, Ramamurthy; Fedoroff, Nina

    2001-01-01

    We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes. PMID:11416215

  11. Programmable and automated bead-based microfluidics for versatile DNA microarrays under isothermal conditions.

    PubMed

    Penchovsky, Robert

    2013-06-21

    Advances in modern genomic research depend heavily on applications of various devices for automated high- or ultra-throughput arrays. Micro- and nanofluidics offer possibilities for miniaturization and integration of many different arrays onto a single device. Therefore, such devices are becoming a platform of choice for developing analytical instruments for modern biotechnology. This paper presents an implementation of a bead-based microfluidic platform for fully automated and programmable DNA microarrays. The devices are designed to work under isothermal conditions as DNA immobilization and hybridization transfer are performed under steady temperature using reversible pH alterations of reaction solutions. This offers the possibility for integration of more selection modules onto a single chip compared to maintaining a temperature gradient. This novel technology allows integration of many modules on a single reusable chip reducing the application cost. The method takes advantage of demonstrated high-speed DNA hybridization kinetics and denaturation on beads under flow conditions, high-fidelity of DNA hybridization, and small sample volumes are needed. The microfluidic devices are applied for a single nucleotide polymorphism analysis and DNA sequencing by synthesis without the need for fluorescent removal step. Apart from that, the microfluidic platform presented is applicable to many areas of modern biotechnology, including biosensor devices, DNA hybridization microarrays, molecular computation, on-chip nucleic acid selection, high-throughput screening of chemical libraries for drug discovery.

  12. DNA microarrays on silicon nanostructures: optimization of the multilayer stack for fluorescence detection.

    PubMed

    Oillic, C; Mur, P; Blanquet, E; Delapierre, G; Vinet, F; Billon, T

    2007-04-15

    To improve the sensitivity of fluorescence detection in DNA microarrays, the use of silicon nanostructures based on chemical vapor deposition (CVD) processes adopted for the growth of rough polycrystalline silicon was investigated. These substrates present advantages of two main properties which could lead to an enhancement of the fluorescence detection, i.e. (i) the increase of the available surface area in order to achieve a high loading capacity of biomolecules and (ii) the optimization of the stack of silicon nanostructures support. Indeed, the structures were elaborated on an initial thermal oxide layer and then covered with a silicon oxide layer, obtained by oxidation and allowing the functionalization for the subsequent grafting of DNA probes. Moreover, these oxide layers play a part in the fluorescence detection. The influence of the silicon oxide layer thickness above and below the silicon grains in close relation with the density of nanostructures on the emitted fluorescence was emphasized. This paper presents an experimental characterization of the fluorescence intensity and the optimization of the different layers that composed the substrate used for DNA microarrays. The performances of the microarrays were investigated by means of hybridization experiments using complementary fluorescent labeled-oligonucleotides targets. Our results indicate that an optimized substrate can be designed and that the use of oxidized silicon nanostructures for support of biochip could be a strategy for improving the sensitivity of fluorescence detection.

  13. MASQOT: a method for cDNA microarray spot quality control

    PubMed Central

    Bylesjö, Max; Eriksson, Daniel; Sjödin, Andreas; Sjöström, Michael; Jansson, Stefan; Antti, Henrik; Trygg, Johan

    2005-01-01

    Background cDNA microarray technology has emerged as a major player in the parallel detection of biomolecules, but still suffers from fundamental technical problems. Identifying and removing unreliable data is crucial to prevent the risk of receiving illusive analysis results. Visual assessment of spot quality is still a common procedure, despite the time-consuming work of manually inspecting spots in the range of hundreds of thousands or more. Results A novel methodology for cDNA microarray spot quality control is outlined. Multivariate discriminant analysis was used to assess spot quality based on existing and novel descriptors. The presented methodology displays high reproducibility and was found superior in identifying unreliable data compared to other evaluated methodologies. Conclusion The proposed methodology for cDNA microarray spot quality control generates non-discrete values of spot quality which can be utilized as weights in subsequent analysis procedures as well as to discard spots of undesired quality using the suggested threshold values. The MASQOT approach provides a consistent assessment of spot quality and can be considered an alternative to the labor-intensive manual quality assessment process. PMID:16223442

  14. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

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

    SciTech Connect

    Mintseris, Julian; Eisen, Michael B.

    2006-07-07

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

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

    PubMed Central

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

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH indicators, which offer many ideal teaching characteristics. The simulation requires no specialized equipment, is very inexpensive, is very reliable, and takes very little preparation time. Student and teacher assessment data indicate the simulation is popular with both groups, and students show significant learning gains. We include many resources with this publication, including all prelab introductory materials (e.g., a paper microarray activity), the student handouts, teachers notes, and pre- and postassessment tools. We did not test the simulation on other student populations, but based on teacher feedback, the simulation also may fit well in community college and in introductory and nonmajors' college biology curricula. PMID:17146040

  17. An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies.

    PubMed

    Adriaens, Michiel E; Jaillard, Magali; Eijssen, Lars M T; Mayer, Claus-Dieter; Evelo, Chris T A

    2012-01-25

    The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization

  18. Microarray gene expression profiling analysis combined with bioinformatics in multiple sclerosis.

    PubMed

    Liu, Mingyuan; Hou, Xiaojun; Zhang, Ping; Hao, Yong; Yang, Yiting; Wu, Xiongfeng; Zhu, Desheng; Guan, Yangtai

    2013-05-01

    Multiple sclerosis (MS) is the most prevalent demyelinating disease and the principal cause of neurological disability in young adults. Recent microarray gene expression profiling studies have identified several genetic variants contributing to the complex pathogenesis of MS, however, expressional and functional studies are still required to further understand its molecular mechanism. The present study aimed to analyze the molecular mechanism of MS using microarray analysis combined with bioinformatics techniques. We downloaded the gene expression profile of MS from Gene Expression Omnibus (GEO) and analysed the microarray data using the differentially coexpressed genes (DCGs) and links package in R and Database for Annotation, Visualization and Integrated Discovery. The regulatory impact factor (RIF) algorithm was used to measure the impact factor of transcription factor. A total of 1,297 DCGs between MS patients and healthy controls were identified. Functional annotation indicated that these DCGs were associated with immune and neurological functions. Furthermore, the RIF result suggested that IKZF1, BACH1, CEBPB, EGR1, FOS may play central regulatory roles in controlling gene expression in the pathogenesis of MS. Our findings confirm the presence of multiple molecular alterations in MS and indicate the possibility for identifying prognostic factors associated with MS pathogenesis.

  19. Nonlinear matching measure for the analysis of on-off type DNA microarray images

    NASA Astrophysics Data System (ADS)

    Kim, Jong D.; Park, Misun; Kim, Jongwon

    2003-07-01

    In this paper, we propose a new nonlinear matching measure for automatic analysis of the on-off type DNA microarray images in which the hybridized spots are detected by the template matching method. The targeting spots of HPV DNA chips are designed for genotyping the human papilloma virus(HPV). The proposed measure is obtained by binarythresholding over the whole template region and taking the number of white pixels inside the spotted area. This measure is evaluated in terms of the accuracy of the estimated marker location to show better performance than the normalized covariance.

  20. Use of microarray technology to profile gene expression patterns important for reproduction in cattle.

    PubMed

    Evans, A C O; Forde, N; O'Gorman, G M; Zielak, A E; Lonergan, P; Fair, T

    2008-07-01

    Fertility in cattle is a major component of many agricultural enterprises and there is pressure to devise methods to improve this. A number of approaches are ongoing, one of which is to better understand the cellular and molecular events of the development of reproductive tissues and to use these as targets for developing new strategies. Microarray technologies now allow us the potential to determine the transcriptional profile of expressed genes in a given tissue. This review focuses on the types of microarrays available for studies in cattle and concludes that genes associated with one or more of the cellular processes of cell survival/death, intracellular signalling, transcription and translation, cell division and proliferation and cellular metabolism are the main transcriptional pathways that control the development of ovarian follicles, oocytes, early embryos and the uterine endometrium about the time of the establishment of pregnancy.

  1. Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups

    PubMed Central

    Guo, Dan; Liu, Bin; Liu, Fenxia; Cao, Boyang; Chen, Min; Hao, Xiyan; Feng, Lu

    2013-01-01

    Salmonella is a major cause of food-borne disease in many countries. Serotype determination of Salmonella is important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46 Salmonella O serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293 Salmonella strains, 186 Shigella strains, representative Escherichia coli strains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of the Salmonella strains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46 Salmonella O serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multiple Salmonella O serogroups simultaneously. PMID:23524674

  2. A hill-climbing approach for automatic gridding of cDNA microarray images.

    PubMed

    Rueda, Luis; Vidyadharan, Vidya

    2006-01-01

    Image and statistical analysis are two important stages of cDNA microarrays. Of these, gridding is necessary to accurately identify the location of each spot while extracting spot intensities from the microarray images and automating this procedure permits high-throughput analysis. Due to the deficiencies of the equipment used to print the arrays, rotations, misalignments, high contamination with noise and artifacts, and the enormous amount of data generated, solving the gridding problem by means of an automatic system is not trivial. Existing techniques to solve the automatic grid segmentation problem cover only limited aspects of this challenging problem and require the user to specify the size of the spots, the number of rows and columns in the grid, and boundary conditions. In this paper, a hill-climbing automatic gridding and spot quantification technique is proposed which takes a microarray image (or a subgrid) as input and makes no assumptions about the size of the spots, rows, and columns in the grid. The proposed method is based on a hill-climbing approach that utilizes different objective functions. The method has been found to effectively detect the grids on microarray images drawn from databases from GEO and the Stanford genomic laboratories.

  3. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    PubMed

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  4. Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

    PubMed

    Palma, Pablo; Cuadros, Marta; Conde-Muíño, Raquel; Olmedo, Carmen; Cano, Carlos; Segura-Jiménez, Inmaculada; Blanco, Armando; Bueno, Pablo; Ferrón, J Antonio; Medina, Pedro

    2013-01-01

    Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders). Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11) and non-responders (n = 16) to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029) gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with locally advanced

  5. Development of a prostate cDNA microarray and statistical gene expression analysis package.

    PubMed

    Carlisle, A J; Prabhu, V V; Elkahloun, A; Hudson, J; Trent, J M; Linehan, W M; Williams, E D; Emmert-Buck, M R; Liotta, L A; Munson, P J; Krizman, D B

    2000-05-01

    A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.

  6. Comparison of hepatocellular carcinoma miRNA expression profiling as evaluated by next generation sequencing and microarray.

    PubMed

    Murakami, Yoshiki; Tanahashi, Toshihito; Okada, Rina; Toyoda, Hidenori; Kumada, Takashi; Enomoto, Masaru; Tamori, Akihiro; Kawada, Norifumi; Taguchi, Y-h; Azuma, Takeshi

    2014-01-01

    MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10(-4), and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.

  7. Microarray technology for the study of DNA damage by low-energy electrons

    NASA Astrophysics Data System (ADS)

    Solomun, T.; Hultschig, C.; Illenberger, E.

    2005-08-01

    The damage induced to a model DNA (dT{25}) immobilized on a gold surface by the interaction of low-energy (1 eV) electrons was studied by means of microarray technology. High quality single-stranded DNA arrays were hybridized with a dye-marked complementary strand after irradiation with electrons and the normalized fluorescence data were used to quantify the DNA damage. The data clearly show the sensitivity of the method. A significant loss of genetic information was already observed at dose as low as few hundred of electrons per immobilized oligonucleotide. The results imply that single stranded DNA and RNA are appreciably more sensitive to radiation and the attack of secondary electrons during replication, transcription or translation stages than the current radiation damage models envisage.

  8. Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

    PubMed Central

    Li, Jiping; Chen, Shu; Evans, David H.

    2001-01-01

    A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods. PMID:11158130

  9. New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

    PubMed

    Pérez-Amador, M A; Lidder, P; Johnson, M A; Landgraf, J; Wisman, E; Green, P J

    2001-12-01

    In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

  10. Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling.

    PubMed

    Nagy, Zsolt B; Kelemen, János Z; Fehér, Liliána Z; Zvara, Agnes; Juhász, Kata; Puskás, László G

    2005-02-01

    Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.

  11. Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

    PubMed

    Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

    2006-07-01

    To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

  12. Genomics of hepatitis B virus-related hepatocellular carcinoma and adjacent noncancerous tissues with cDNA microarray.

    PubMed

    Huang, Yu-kun; Fan, Xue-gong; Qiu, Fu; Wang, Zhi-ming

    2011-07-05

    Hepatocellular carcinoma (HCC) is a common primary cancer frequently associated with hepatitis B virus (HBV) infection. However, whether these identified genes are particularly associated with HBV-related HCC remains unknown. The aim of this study was to investigate the differential gene expression between HBV-related HCC tissues and adjacent noncancerous tissues. cDNA microarray was used to detect the differential gene expression profile in the HBV-related HCC tissues and adjacent noncancerous tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the differential expression of candidate genes obtained from cDNA microarray experiment. In this study, 1369 genes or expressed sequence tags (ESTs) including 121 genes or ESTs with at least two-fold expression alterations between cancerous and noncancerous tissues were identified. Special AT-rich sequence binding protein 1 (SATB-1) expression was positive in 73% (16/22) of cancerous tissues and negative (0/22) in all noncancerous tissues of HBV-related HCC patients. Transmembrane 4 superfamily member 1 (TM4SF-1) expression was positive in 86% (19/22) of cancerous tissues and negative (0/22) in all noncancerous tissues. Suppression of tumorigenicity 14 (ST-14) expression was positive in 73% (16/22) of noncancerous tissues in patients with HBV-related HCC and negative in all HCC tissues (0/22). This study provided the gene expression profile of HBV-related HCC and presented differential expression patterns of SATB-1, TM4SF-1 and ST-14 between cancerous and noncancerous tissues in patients with HBV-related HCC.

  13. Simple PCR-based DNA microarray system to identify human pathogenic fungi in skin.

    PubMed

    Sato, Tomotaka; Takayanagi, Atsushi; Nagao, Keisuke; Tomatsu, Nobuhiro; Fukui, Toshifumi; Kawaguchi, Masahiro; Kudoh, Jun; Amagai, Masayuki; Yamamoto, Nobuko; Shimizu, Nobuyoshi

    2010-07-01

    Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

  14. Specific mutation screening of TP53 gene by low-density DNA microarray

    PubMed Central

    Rangel-López, Angélica; Méndez-Tenorio, Alfonso; Beattie, Kenneth L; Maldonado, Rogelio; Mendoza, Patricia; Vázquez, Guelaguetza; Pérez-Plasencia, Carlos; Sánchez, Martha; Navarro, Guillermo; Salcedo, Mauricio

    2009-01-01

    TP53 is the most commonly mutated gene in human cancers. Approximately 90% of mutations in this gene are localized between domains encoding exons 5 to 8. The aim of this investigation was to examine the ability of the low density DNA microarray with the assistance of double tandem hybridization platform to characterize TP53 mutational hotspots in exons 5, 7, and 8 of the TP53. Nineteen capture probes specific to each potential mutation site were designed to hybridize to specific site. Virtual hybridization was used to predict the stability of hybridization of each capture probe with the target. Thirty-three DNA samples from different sources were analyzed for mutants in these exons. A total of 32 codon substitutions were found by DNA sequencing. 24 of them a showed a perfect correlation with the hybridization pattern system and DNA sequencing analysis of the regions scanned. Although in this work we directed our attention to some of the most representative mutations of the TP53 gene, the results suggest that this microarray system proved to be a rapid, reliable, and effective method for screening all the mutations in TP53 gene. PMID:24198462

  15. Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

    PubMed Central

    Schwartz, David Eric; Gong, Ping; Shepard, Kenneth L.

    2008-01-01

    We present an active oligonucleotide microarray platform for time-resolved Förster resonance energy transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors. PMID:18515059

  16. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences

    PubMed Central

    Moran, Sebastian; Arribas, Carles; Esteller, Manel

    2016-01-01

    Aim: DNA methylation is the best known epigenetic mark. Cancer and other pathologies show an altered DNA methylome. However, delivering complete DNA methylation maps is compromised by the price and labor-intensive interpretation of single nucleotide methods. Material & methods: Following the success of the HumanMethylation450 BeadChip (Infinium) methylation microarray (450K), we report the technical and biological validation of the newly developed MethylationEPIC BeadChip (Infinium) microarray that covers over 850,000 CpG methylation sites (850K). The 850K microarray contains >90% of the 450K sites, but adds 333,265 CpGs located in enhancer regions identified by the ENCODE and FANTOM5 projects. Results & conclusion: The 850K array demonstrates high reproducibility at the 450K CpG sites, is consistent among technical replicates, is reliable in the matched study of fresh frozen versus formalin-fixed paraffin-embeded samples and is also useful for 5-hydroxymethylcytosine. These results highlight the value of the MethylationEPIC BeadChip as a useful tool for the analysis of the DNA methylation profile of the human genome. PMID:26673039

  17. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences.

    PubMed

    Moran, Sebastian; Arribas, Carles; Esteller, Manel

    2016-03-01

    DNA methylation is the best known epigenetic mark. Cancer and other pathologies show an altered DNA methylome. However, delivering complete DNA methylation maps is compromised by the price and labor-intensive interpretation of single nucleotide methods. Following the success of the HumanMethylation450 BeadChip (Infinium) methylation microarray (450K), we report the technical and biological validation of the newly developed MethylationEPIC BeadChip (Infinium) microarray that covers over 850,000 CpG methylation sites (850K). The 850K microarray contains >90% of the 450K sites, but adds 333,265 CpGs located in enhancer regions identified by the ENCODE and FANTOM5 projects. The 850K array demonstrates high reproducibility at the 450K CpG sites, is consistent among technical replicates, is reliable in the matched study of fresh frozen versus formalin-fixed paraffin-embeded samples and is also useful for 5-hydroxymethylcytosine. These results highlight the value of the MethylationEPIC BeadChip as a useful tool for the analysis of the DNA methylation profile of the human genome.

  18. Profiling the Humoral Immune Response of Acute and Chronic Q Fever by Protein Microarray*

    PubMed Central

    Vigil, Adam; Chen, Chen; Jain, Aarti; Nakajima-Sasaki, Rie; Jasinskas, Algimantas; Pablo, Jozelyn; Hendrix, Laura R.; Samuel, James E.; Felgner, Philip L.

    2011-01-01

    Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response. PMID:21817167

  19. Fully integrated miniature device for automated gene expression DNA microarray processing.

    PubMed

    Liu, Robin Hui; Nguyen, Tai; Schwarzkopf, Kevin; Fuji, H Sho; Petrova, Alla; Siuda, Tony; Peyvan, Kia; Bizak, Michael; Danley, David; McShea, Andy

    2006-03-15

    A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.

  20. Fabrication of DNA microarrays on nanoengineered polymericultrathin film prepared by self-assembly of polyelectrolytemultilayers

    SciTech Connect

    Zhou, X.; Wu Liyou; Zhou, J.-Z.

    2004-08-24

    Microarray-based technology is in need of flexible andcost-effective chemistry for fabrication of oligonucleotide microarrays.We have developed a novel method for the fabrication of oligonucleotidemicroarrays with unmodified oligonucleotide probes on nanoengineeredthree-dimensional thin films that are deposited on glass slides byconsecutive layer-to-layer adsorption of polyelectrolytes. Unmodifiedoligonucleotide probes were spotted and immobilized on these multilayeredpolyelectrolyte thin films (PET) by electrostatic adsorption andentrapment on the porous structure of the PET film. The PET provideshigher probe binding capacity and thus higher hybridization signal thanthat of the traditional two-dimensional aminosilane and poly-L-lysinecoated slides. Immobilized probe densities of 3.4 x 1012/cm2 wereobserved for microarray spots on PET with unmodified 50-meroligonucleotide probes, which is comparable to the immobilized probedensities of alkyamine-modified 50-mer probes end-tethered on analdehyde-functionalized slide. The study of hybridization efficiencyshowed that 90 percent of immobilized probes on PET film are accessibleto target DNA to form duplex format in hybridization. The DNA microarrayfabricated on PET film has wider dynamic range (about 3 orders ofmagnitude) and lower detection limit (0.5 nM) than the conventionalamino- and aldehyde-functionlized slides. Oligonucleotide microarraysfabricated on these PET-coated slides also had consistent spotmorphology. In addition, discrimination of single nucleotide polymorphismof 16S rRNA genes was achieved with the PET-based oligonucleotidemicroarrays. The PET microarrays constructed by our self-assembly processis cost-effective, versatile, and well suited for immobilizing many typesof biological active molecules so that a wide variety of microarrayformats can be developed.

  1. Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy.

    PubMed

    Zandian, Arash; Forsström, Björn; Häggmark-Månberg, Anna; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter; Ayoglu, Burcu

    2017-02-09

    The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide microarrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

  2. Touch DNA-The prospect of DNA profiles from cables.

    PubMed

    Lim, Sharon; Subhani, Zuhaib; Daniel, Barbara; Frascione, Nunzianda

    2016-05-01

    Metal theft in the railroad industry poses significant challenges to transport investigators. Cable sheaths left behind at crime scenes, if appropriately analysed, could provide valuable evidence in a forensic investigation, but attempts at recovering DNA are not routinely made. Experiments were set up to ascertain the success in DNA recovery from the surface of cable sheaths after deposition of (a) sweat, (b) extracted DNA and (c) fingermarks. Since investigators try to collect fingermarks and often treat the cables with cyanoacrylate fuming (CNA fuming) or wet powder suspensions (WPS) to enhance the marks this study investigated the recovery of DNA from fingermarks pre- and post-enhancement. The double-swab technique and mini-taping were compared as options to recover DNA from the cable sheaths. Results demonstrate that generally, there is no significant difference between using swabs or mini-tapes to recover the DNA from the non-porous cables (p>0.05). It was also illustrated that CNA fuming performed better than WPS in terms of subsequent recovery and profiling of DNA. CNA fuming resulted in an average increase in DNA recovered via swabbing and taping (more than 4× and 8×, respectively), as compared to no treatment, with 50% of the DNA recovered after CNA fuming generating full DNA profiles. Copyright © 2016 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.

  3. Mechanisms Underlying the Antiproliferative and Prodifferentiative Effects of Psoralen on Adult Neural Stem Cells via DNA Microarray

    PubMed Central

    Ning, You; Huang, Jian-Hua; Xia, Shi-Jin; Bian, Qin; Chen, Yang; Zhang, Xin-Min; Dong, Jing-Cheng; Shen, Zi-Yin

    2013-01-01

    Adult neural stem cells (NSCs) persist throughout life to replace mature cells that are lost during turnover, disease, or injury. The investigation of NSC creates novel treatments for central nervous system (CNS) injuries and neurodegenerative disorders. The plasticity and reparative potential of NSC are regulated by different factors, which are critical for neurological regenerative medicine research. We investigated the effects of Psoralen, which is the mature fruit of Psoralea corylifolia L., on NSC behaviors and the underlying mechanisms. The self-renewal and proliferation of NSC were examined. We detected neuron- and/or astrocyte-specific markers using immunofluorescence and Western blotting, which could evaluate NSC differentiation. Psoralen treatment significantly inhibited neurosphere formation in a dose-dependent manner. Psoralen treatment increased the expression of the astrocyte-specific marker but decreased neuron-specific marker expression. These results suggested that Psoralen was a differentiation inducer in astrocyte. Differential gene expression following Psoralen treatment was screened using DNA microarray and confirmed by quantitative real-time PCR. Our microarray study demonstrated that Psoralen could effectively regulate the specific gene expression profile of NSC. The genes involved in the classification of cellular differentiation, proliferation, and metabolism, the transcription factors belonging to Ets family, and the hedgehog pathway may be closely related to the regulation. PMID:23983781

  4. Scanning electrochemical microscopy of DNA hybridization on DNA microarrays enhanced by HRP-modified SiO2 nanoparticles.

    PubMed

    Fan, Huajun; Wang, Xiaolan; Jiao, Fang; Zhang, Fan; Wang, Qingjiang; He, Pingang; Fang, Yuzhi

    2013-07-02

    Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

  5. DNA methylation profiling of hematopoietic stem cells.

    PubMed

    Begtrup, Amber Hogart

    2014-01-01

    DNA methylation is a key epigenetic mark that is essential for properly functioning hematopoietic stem cells. Determining where functionally relevant DNA methylation marks exist in the genome is crucial to understanding the role that methylation plays in hematopoiesis. This chapter describes a method to profile DNA methylation by selectively enriching methylated DNA sequences that are bound in vitro by methyl-binding domain (MBD) proteins. The MBD-pulldown approach selects for DNA sequences that have the potential to be "read" by the endogenous machinery involved in epigenetic regulation. Furthermore, this approach is feasible with very small quantities of DNA, and is compatible with the use of any downstream high-throughput sequencing approach. This technique offers a reliable, simple, and powerful tool for exploration of the role of DNA methylation in hematopoietic stem cells.

  6. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    PubMed

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  7. Long non-coding RNA expression profiles in gallbladder carcinoma identified using microarray analysis

    PubMed Central

    Wang, Jiwen; Liu, Han; Shen, Xiaokun; Wang, Yueqi; Zhang, Dexiang; Shen, Sheng; Suo, Tao; Pan, Hongtao; Ming, Yue; Ding, Kan; Liu, Houbao

    2017-01-01

    Gallbladder carcinoma (GBC) is the most common biliary tract cancer and exhibits poor patient prognosis. Previous studies have identified that long non-coding RNAs (lncRNAs) serve important regulatory roles in cancer biology. Alterations in lncRNAs are associated with several types of cancer. However, the contribution of lncRNAs to GBC remains unclear. To investigate the lncRNAs that are potentially involved in GBC, lncRNA profiles were identified in three pairs of human GBC and corresponding peri-carcinomatous tissue samples using microarray analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to validate the microarray data. In order to elucidate potential functions, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes analysis, and network analysis were used to determine relevant signaling pathways. Abundant RNA probes were used, and 1,758 lncRNAs and 1,254 mRNAs were detected to be differentially expressed by the microarray. Compared with para-carcinoma tissue, numerous lncRNAs were markedly upregulated or downregulated in GBC. The results demonstrated that the lncRNAs that were downregulated in GBC were more numerous compared with the lncRNAs that were upregulated. Among them, RP11-152P17.2-006 was the most upregulated, whereas CTA-941F9.9 was the most downregulated. The RT-qPCR results were consistent with the microarray data. Pathway analysis indicated that five pathways corresponded to the differentially expressed transcripts. It was demonstrated that lncRNA expression in GBC was markedly altered, and a series of novel lncRNAs associated with GBC were identified. The results of the present study suggest that the functions of lncRNAs are important in GBC development and progression. PMID:28529578

  8. Long non-coding RNA expression profiles in gallbladder carcinoma identified using microarray analysis.

    PubMed

    Wang, Jiwen; Liu, Han; Shen, Xiaokun; Wang, Yueqi; Zhang, Dexiang; Shen, Sheng; Suo, Tao; Pan, Hongtao; Ming, Yue; Ding, Kan; Liu, Houbao

    2017-05-01

    Gallbladder carcinoma (GBC) is the most common biliary tract cancer and exhibits poor patient prognosis. Previous studies have identified that long non-coding RNAs (lncRNAs) serve important regulatory roles in cancer biology. Alterations in lncRNAs are associated with several types of cancer. However, the contribution of lncRNAs to GBC remains unclear. To investigate the lncRNAs that are potentially involved in GBC, lncRNA profiles were identified in three pairs of human GBC and corresponding peri-carcinomatous tissue samples using microarray analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to validate the microarray data. In order to elucidate potential functions, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes analysis, and network analysis were used to determine relevant signaling pathways. Abundant RNA probes were used, and 1,758 lncRNAs and 1,254 mRNAs were detected to be differentially expressed by the microarray. Compared with para-carcinoma tissue, numerous lncRNAs were markedly upregulated or downregulated in GBC. The results demonstrated that the lncRNAs that were downregulated in GBC were more numerous compared with the lncRNAs that were upregulated. Among them, RP11-152P17.2-006 was the most upregulated, whereas CTA-941F9.9 was the most downregulated. The RT-qPCR results were consistent with the microarray data. Pathway analysis indicated that five pathways corresponded to the differentially expressed transcripts. It was demonstrated that lncRNA expression in GBC was markedly altered, and a series of novel lncRNAs associated with GBC were identified. The results of the present study suggest that the functions of lncRNAs are important in GBC development and progression.

  9. DNA profiling from heroin street dose packages.

    PubMed

    Zamir, Ashira; Cohen, Yaron; Azoury, Myriam

    2007-03-01

    A large amount of heroin street doses are seized and examined for drug content by the Israel police. These are generally wrapped in heat-sealed plastic. Occasionally it is possible to visualize latent fingerprints on the plastic wrap itself, but the small size of the plastic item and the sealing process makes the success rate very low. In this study, the possibility of extracting and profiling DNA from the burnt edge of the plastic wrap was investigated. The idea was based on the assumption that epithelial cells might be trapped during the sealing process. The results show that there are sufficient quantities of DNA deposited at the "amorphic" burnt edges of sealed street doses for DNA profiling to be carried out. A controlled experiment using a known donor was performed. This subject carried out sealing of "street drug" packages and consequent DNA extractions were performed to show that known DNA profiles could be recovered from such packages, as a result of handling by the "packer." "Square-like" burnt edges did not yield DNA profiles, probably because of differences in the sealing process. It was also shown that DNA could be recovered from the plastic wrap itself and not only from the amorphic burnt edges. As heroin dealers and drug users are often involved in other crimes and run-ins with the law, the effective extraction and addition of their DNA profiles from such items of evidence to the newly established DNA database in Israel provides new avenues in the continued fight against crime and drug traffickers.

  10. Integration of clinical point-of-care requirements in a DNA microarray genotyping test.

    PubMed

    Van Dorst, Bieke; Cremers, Amelieke; Jans, Karolien; Van Domburg, Trees; Steegen, Kim; Huang, Chengjun; Dorrer, Christian; Lagae, Liesbet; Ferwerda, Gerben; Stuyver, Lieven J

    2014-11-15

    Various proof-of-concept studies have shown the potential of biosensors with a high multiplex detection capability for the readout of DNA microarrays in a lab-on-a-chip. This is particularly interesting for the development of point-of-care genotyping tests, to screen for multiple pathogens and/or antibiotic resistance patterns. In this paper, an assay workflow is presented, suited for the development of novel lab-on-a-chips with an integrated DNA microarray. Besides the description of the different assay steps (DNA purification, amplification and detection), a control strategy is presented according to recommendations of the US Food and Drug Administration (FDA). To use a lab-on-a-chip for diagnostic applications, the optimization and evaluation of the assay performance with clinical samples is very important. Therefore, appropriate quantification methods are described, which allow optimization and evaluation of the separate assay steps, as well as total assay performance. In order to demonstrate and evaluate the total workflow, blood samples spiked with Streptococcus pneumoniae were tested. All blood samples with ≥ 10(3)CFU S. pneumoniae per ml of human blood were successfully detected by this genotyping assay.

  11. Metatranscriptomic analysis of the response of river biofilms to pharmaceutical products, using anonymous DNA microarrays.

    PubMed

    Yergeau, Etienne; Lawrence, John R; Waiser, Marley J; Korber, Darren R; Greer, Charles W

    2010-08-01

    Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to microg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems.

  12. Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.

    PubMed

    Raghavachari, Nalini; Bao, Yijia P; Li, Guangshan; Xie, Xinying; Müller, Uwe R

    2003-01-15

    Microarray technology is currently being used extensively in functional genomics research and modern drug discovery and development. Henceforward, tremendous application potential for this technology exists in the fields of clinical diagnostics and prognostics, pathology, and toxicology for high-throughput analysis of "disease" gene expression. However, the major hurdle now in this technology is not the performance of the arrays but rather the efficient reproducibility of the hybridization signal intensity in a fluorescence-based analysis. The sensitivity of fluorescence detection on an array is to a large extent limited by the amount of background signal arising due to nonspecifically bound probes and fluorescence that is intrinsically associated with the chip substrate and/or the attached target DNA, the so-called autofluorescence. Here, we describe a simple and efficient method to reduce autofluorescence from undetermined sources on coated glass slides with and without DNA arrays. This sodium borohydride-mediated reduction process resulted in significantly lower and more even background fluorescence. This in turn extended the dynamic range of detection and reduced the average coefficient of variation of fluorescent signal ratios on DNA microarrays in addition to improving the detection of genes that are expressed at a low level.

  13. Shrink-Induced Silica Multiscale Structures for Enhanced Fluorescence from DNA Microarrays

    PubMed Central

    2015-01-01

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30–45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays. PMID:25191785

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

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

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

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

    PubMed

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

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

  16. Characterization, validation and application of a DNA microarray for the detection of mandatory and other waterborne pathogens.

    PubMed

    Gomes, Maria; Vieira, Helena; Vale, Filipa F

    2015-11-01

    Culture methods for the detection of indicator bacteria are currently used for detection of waterborne bacteria. The need for an increased range of analyzed bacteria coupled with the obtainment of rapid and early results justify the development of a DNA microarray for the identification of waterborne pathogens. This DNA microarray has 16 implanted probes with a median size of 147 bases, targeting 12 different parameters, including all mandatory indicator microorganisms, such as Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus, total and fecal coliforms and enterococci. The validation performed with DNA extracted from pure microbial cultures showed the suitability of the probes for detection of the target microorganism. To overcome the high dilution of water samples it was included either a prior culture step of bacterial contaminants retained after filtering 100 ml of water, or a 10-fold increase in the volume of filtered water, that resulted in the increase of the detected bacteria. The analysis of complex environmental water samples using culture methods and the DNA microarray revealed that the latter detected the same parameters plus other bacteria tested only in the DNA microarray. The results show that this DNA microarray may be a useful tool for water microbiological surveillance. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  17. Microarray profile of differentially expressed genes in a monkey model of allergic asthma

    PubMed Central

    Zou, Jun; Young, Simon; Zhu, Feng; Gheyas, Ferdous; Skeans, Susan; Wan, Yuntao; Wang, Luquan; Ding, Wei; Billah, Motasim; McClanahan, Terri; Coffman, Robert L; Egan, Robert; Umland, Shelby

    2002-01-01

    Background Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs. Results Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only. Conclusion This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model. PMID:12049661

  18. Verification of gene expression profiles for colorectal cancer using 12 internet public microarray datasets

    PubMed Central

    Chang, Yu-Tien; Yao, Chung-Tay; Su, Sui-Lung; Chou, Yu-Ching; Chu, Chi-Ming; Huang, Chi-Shuan; Terng, Harn-Jing; Chou, Hsiu-Ling; Wetter, Thomas; Chen, Kang-Hua; Chang, Chi-Wen; Shih, Yun-Wen; Lai, Ching-Huang

    2014-01-01

    AIM: To verify gene expression profiles for colorectal cancer using 12 internet public microarray datasets. METHODS: Logistic regression analysis was performed, and odds ratios for each gene were determined between colorectal cancer (CRC) and controls. Twelve public microarray datasets of GSE 4107, 4183, 8671, 9348, 10961, 13067, 13294, 13471, 14333, 15960, 17538, and 18105, which included 519 cases of adenocarcinoma and 88 normal mucosa controls, were pooled and used to verify 17 selective genes from 3 published studies and estimate the external generality. RESULTS: We validated the 17 CRC-associated genes from studies by Chang et al (Model 1: 5 genes), Marshall et al (Model 2: 7 genes) and Han et al (Model 3: 5 genes) and performed the multivariate logistic regression analysis using the pooled 12 public microarray datasets as well as the external validation. The goodness-of-fit test of Hosmer-Lemeshow (H-L) showed statistical significance (P = 0.044) for Model 2 of Marshall et al in which observed event rates did not match expected event rates in subgroups of the model population. Expected and observed event rates in subgroups were similar, which are called well calibrated, in Models 1, 3 and 4 with non-significant P values of 0.460, 0.194 and 1.000 for H-L tests, respectively. A 7-gene model of CPEB4, EIF2S3, MGC20553, MS4A1, ANXA3, TNFAIP6 and IL2RB was pairwise selected, which showed the best results in logistic regression analysis (H-L P = 1.000, R2 = 0.951, areas under the curve = 0.999, accuracy = 0.968, specificity = 0.966 and sensitivity = 0.994). CONCLUSION: A novel gene expression profile was associated with CRC and can potentially be applied to blood-based detection assays. PMID:25516661

  19. D-MaPs - DNA-microarray projects: Web-based software for multi-platform microarray analysis

    PubMed Central

    2009-01-01

    The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core. PMID:21637530

  20. D-MaPs - DNA-microarray projects: Web-based software for multi-platform microarray analysis.

    PubMed

    Carazzolle, Marcelo F; Herig, Taís S; Deckmann, Ana C; Pereira, Gonçalo A G

    2009-07-01

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

  1. Thymus and Myasthenia Gravis: what can we learn from DNA microarrays?

    PubMed

    Cizeron-Clairac, Géraldine; Le Panse, Rozen; Frenkian-Cuvelier, Mélinée; Meraouna, Amel; Truffault, Frédérique; Bismuth, Jacky; Mussot, Sacha; Kerlero de Rosbo, Nicole; Berrih-Aknin, Sonia

    2008-09-15

    This review is dedicated to John Newsom-Davis, who was an exceptional colleague and friend, always exchanging ideas with respect and consideration. We shall not forget his involvement and passion in search for the truth on the role of thymectomy in the management of Myasthenia Gravis (MG). In this short review, we shall summarize what we learnt from DNA microarrays applied to MG thymus. We shall focus on three main comparisons of the thymic transcriptomes: 1) highly hyperplastic MG patients versus non-MG adults; 2) corticosteroid-treated versus untreated seropositive MG patients; and 3) seronegative versus seropositive MG patients.

  2. DNA microarray-based detection of multiple pathogens: Mycoplasma spp. and Chlamydia spp.

    PubMed

    Schnee, Christiane; Sachse, Konrad

    2015-01-01

    Rapid detection of slow-growing or non-culturable microorganisms, such as Mycoplasma spp. and Chlamydia spp., is still a challenge to diagnosticians in the veterinary field. In addition, as epidemiological evidence on the frequency of mixed infections involving two and more bacterial species has been emerging, detection methods allowing simultaneous identification of different pathogens are required. In the present chapter, we describe DNA microarray-based procedures for the detection of 83 Mollicutes species (Mycoplasma assay) and 11 Chlamydia spp. (Chlamydia assay). The assays are suitable for use in a routine diagnostic environment, as well as in microbiological research.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2006-01-01

    Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers. PMID:16517630

  5. Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance

    PubMed Central

    Grimm, Verena; Ezaki, Satoshi; Susa, Milorad; Knabbe, Cornelius; Schmid, Rolf. D.; Bachmann, Till T.

    2004-01-01

    Standard clinical procedures for pathogen resistance identification are laborious and usually require 2 days of cultivation before the resistance can be determined unequivocally. In contrast, clinicians and patients face increasing threats from antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels of resistance. A major class of microbial resistance stems from the occurrence of beta-lactamases, which, if mutated, can cause the severe extended-spectrum beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase inhibitors. We describe an oligonucleotide microarray for identification of the single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants described to date which are related to the ESBL and/or IRT phenotype. The target DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae cells isolated from clinical samples, was amplified and fluorescently labeled by PCR with consensus primers in the presence of cyanine 5-labeled nucleotides. The total assay, including PCR, hybridization, and image analysis, could be performed in 3.5 h. The microarray results were validated by standard clinical procedures. The microarray outperformed the standard procedures in terms of assay time and the depth of information provided. In conclusion, this array offers an attractive option for the identification and epidemiologic monitoring of TEM beta-lactamases in the routine clinical diagnostic laboratory. PMID:15297528

  6. Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray

    PubMed Central

    Morse, David L.; Balagurunathan, Yoga; Hostetter, Galen; Trissal, Maria; Tafreshi, Narges K.; Burke, Nancy; Lloyd, Mark; Enkemann, Steven; Coppola, Domenico; Hruby, Victor; Gillies, Robert J.; Han, Haiyong

    2010-01-01

    Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer. PMID:20510208

  7. Aberrant Expression Profile of Long Noncoding RNA in Human Sinonasal Squamous Cell Carcinoma by Microarray Analysis

    PubMed Central

    Meng, Ling-zhao; Sun, Jing-wu; Yang, Fan

    2016-01-01

    Objectives. This study aimed to identify aberrantly expressed long noncoding RNAs (lncRNAs) profile of sinonasal squamous cell carcinoma (SSCC) and explore their potential functions. Methods. We investigated lncRNA and mRNA expression in SSCC and paired adjacent noncancerous tissues obtained from 6 patients with microarrays. Gene ontology (GO) analysis and pathway analysis were utilized to investigate the gene function. Gene signal-network and lncRNA-mRNA network were depicted. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to validate 5 lncRNAs in a second set of paired SSCC and adjacent noncancerous tissues obtained from 22 additional patients. Results. We identified significantly differentially expressed lncRNAs (n = 3146) and mRNAs (n = 2208) in SSCC relative to noncancerous tissues. The GO annotation indicated that there are some core gene products that may be attributed to the progress of SSCC. The pathway analysis identified many pathways associated with cancer. The results of lncRNA-mRNA network and gene signal-network implied some core lncRNAs/mRNAs might play important roles in SSCC pathogenesis. The results of qRT-PCR showed that all of the 5 lncRNAs were differentially expressed and consistent with the microarray results. Conclusion. Our study is the first screening and analysis of lncRNAs expression profile in SSCC and may offer new insights into pathogenesis of this disease. PMID:28044124

  8. Profiling Humoral Immune Responses to P. falciparum Infection with Protein Microarrays

    PubMed Central

    Doolan, Denise L.; Mu, Yunxiang; Unal, Berkay; Sundaresh, Suman; Hirst, Siddiqua; Valdez, Conrad; Randall, Arlo; Molina, Douglas; Liang, Xiaowu; Freilich, Daniel A.; Oloo, J. Aggrey; Blair, Peter L.; Aguiar, Joao C.; Baldi, Pierre; Davies, D. Huw; Felgner, Philip L.

    2010-01-01

    A complete description of the serological response following exposure of humans to complex pathogens is lacking and approaches suitable for accomplishing this are limited. Here we report, using malaria as a model, a method which elucidates the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms, and which identifies immunoreactive antigens of interest for vaccine development or other applications. Expression vectors encoding 250 Plasmodium falciparum (Pf) proteins were generated by PCR/recombination cloning; the proteins were individually expressed with >90% efficiency in E. coli cell-free in vitro transcription and translation reactions, and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from one of four groups which differed in immune status: sterile immunity or no immunity against experimental challenge following vaccination with radiation-attenuated Pf sporozoites, partial immunity acquired by natural exposure, and no previous exposure to Pf. Overall, 72 highly reactive Pf antigens were identified. Proteomic features associated with immunoreactivity were identified. Importantly, antibody profiles were distinct for each donor group. Information obtained from such analyses will facilitate identifying antigens for vaccine development, dissecting the molecular basis of immunity, monitoring the outcome of whole-organism vaccine trials, and identifying immune correlates of protection. PMID:18937256

  9. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    PubMed

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  10. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation

    PubMed Central

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi’s individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  11. Optical and surface analysis of DNA microarrays to assess printed spot heterogeneity

    NASA Astrophysics Data System (ADS)

    Nagaraja Rao, Archana

    DNA microarrays have been plagued with analytical problems with quantitation, metrics, figures of merit, and reliability and reproducibility issues, hindering their acceptance in clinical and diagnostic settings. The main deficiency in the printed DNA format is the microspot heterogeneity occurring during array fabrication and further amplified during target hybridization. Work described in this dissertation focuses on assessment of DNA microarray spots generated with conventional pin-type contact printing of fluorescently labeled DNA probes, on industry-standard commercial polymer-coated array slides and their hybridization with complementary oligomer DNA target. Printing of probe DNA microspots shares many features of commonly reported droplet evaporation dynamics that lead to different drying patterns and spot morphologies. This study directly identifies and analyzes different DNA probe chemical and spatial microenvironments within spots, analyzed with high-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical imaging, confocal epifluorescence, and probe microscopy force imaging methods. Drying of DNA probe spots shows Marangoni flow effects with high densities of probe DNA-Cy3 located in spot centers and nonhomogeneous DNA distributed radially within printed spots with both TOF-SIMS imaging and epifluorescence microscopy. Target hybridization kinetics and duplex formation were assessed using real-time in situ confocal imaging, and confirmed radial hemispherical diffusion-mediated distribution of target capture from spot edge to its interior. Kinetic modeling indicates pseudo-first order kinetics due to transport limitations and local density-dependent probe interactions with diffusing target. Fluorescence resonance energy transfer (FRET) and photobleaching results show that the high- density probe overcrowding in spots facilitates a broad range of target binding interactions regardless of dye orientations. Moreover, lateral probe density

  12. Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  13. Differences in the expression of genes between normal tissue and squamous cell carcinomas of head and neck using cancer-related gene cDNA microarray.

    PubMed

    Kainuma, Kazuyuki; Katsuno, Satoshi; Hashimoto, Shigenari; Oguchi, Tomohiro; Suzuki, Nobuyoshi; Asamura, Kenji; Usami, Shin-ichi

    2006-09-01

    This study clearly showed the molecular characteristics of head and neck squamous cell carcinoma (HNSCC) on the basis of gene expression patterns. cDNA microarray has recently been shown to have the ability to represent the expression patterns of large numbers of genes from a small amount of tissue, potentially enabling definition of groups of patients with similar biological behavior of cancer. Although gene expression profiling using this technique has proven helpful for predicting the prognosis in various cancers, little is known regarding HNSCC. The aim of this study was to investigate the differences in the expression of various genes between normal tissue and cancers of patients with HNSCC by cDNA microarray. We extracted mRNA from 17 HNSCC patients and used cDNA microarray analysis to investigate the gene expression patterns. The present study was not designed to perform an inclusive search for genes but rather to focus on cancer-related genes. Seven independent genes were found to be up-regulated in cancer tissues: matrix metalloproteinase-1, -3, and -10, interleukin-8, cadherin 3, hexabrachion, and interferon gamma-inducible protein 10. Hyaluronic acid-binding protein 2, keratin 4, and keratin 13 were categorized as down-regulated. The hierarchical clustering and dendrogram for 17 cancer samples and 425 genes could be grouped into three clusters.

  14. Development of a microarray platform for FFPET profiling: application to the classification of human tumors

    PubMed Central

    Duenwald, Sven; Zhou, Mingjie; Wang, Yanqun; Lejnine, Serguei; Kulkarni, Amit; Graves, Jaime; Smith, Ryan; Castle, John; Tokiwa, George; Fine, Bernard; Dai, Hongyue; Fare, Thomas; Marton, Matthew

    2009-01-01

    Background mRNA profiling has become an important tool for developing and validating prognostic assays predictive of disease treatment response and outcome. Archives of annotated formalin-fixed paraffin-embedded tissues (FFPET) are available as a potential source for retrospective studies. Methods are needed to profile these FFPET samples that are linked to clinical outcomes to generate hypotheses that could lead to classifiers for clinical applications. Methods We developed a two-color microarray-based profiling platform by optimizing target amplification, experimental design, quality control, and microarray content and applied it to the profiling of FFPET samples. We profiled a set of 50 fresh frozen (FF) breast cancer samples and assigned class labels according to the signature and method by van 't Veer et al [1] and then profiled 50 matched FFPET samples to test how well the FFPET data predicted the class labels. We also compared the sorting power of classifiers derived from FFPET sample data with classifiers derived from data from matched FF samples. Results When a classifier developed with matched FF samples was applied to FFPET data to assign samples to either "good" or "poor" outcome class labels, the classifier was able to assign the FFPET samples to the correct class label with an average error rate = 12% to 16%, respectively, with an Odds Ratio = 36.4 to 60.4, respectively. A classifier derived from FFPET data was able to predict the class label in FFPET samples (leave-one-out cross validation) with an error rate of ~14% (p-value = 3.7 × 10-7). When applied to the matched FF samples, the FFPET-derived classifier was able to assign FF samples to the correct class labels with 96% accuracy. The single misclassification was attributed to poor sample quality, as measured by qPCR on total RNA, which emphasizes the need for sample quality control before profiling. Conclusion We have optimized a platform for expression analyses and have shown that our profiling

  15. A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data

    PubMed Central

    van Hijum, Sacha AFT; de Jong, Anne; Baerends, Richard JS; Karsens, Harma A; Kramer, Naomi E; Larsen, Rasmus; den Hengst, Chris D; Albers, Casper J; Kok, Jan; Kuipers, Oscar P

    2005-01-01

    Background In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed. Results A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance. Conclusion Even in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality. PMID:15907200

  16. Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays.

    PubMed

    Todman, M G; Han, S-K; Herbison, A E

    2005-01-01

    The definition of neurotransmitter receptors expressed by individual neuronal phenotypes is essential for our understanding of integrated neural regulation. We report here a single-neuron strategy using green fluorescent protein (GFP)-promoter transgenic mice and oligonucleotide microarrays that has enabled us to provide a qualitative profile of the neurotransmitter receptors expressed by the gonadotropin- releasing hormone (GnRH) neurons, critical for the neural regulation of fertility. Acute brain slices were prepared from adult female GnRH-GFP transgenic mice and single GnRH neurons identified and patched. The contents of GnRH neurons underwent reverse transcription and cDNA amplification using the switch mechanism at the 5' end of RNA templates system, and hybridization to mouse gene oligonucleotide arrays. Fifty different neurotransmitter receptor subunit mRNAs were detected in GnRH neurons. Many of the classical amino acid and aminergic receptors were present in addition to 14 distinct, and in most cases novel, neuropeptidergic receptor signaling families. Four of the latter were selected for functional validation with gramicidin-perforated patch-clamp electrophysiology. Galanin, GnRH and neuromedin B were all found to exert direct depolarizing actions upon GnRH neurons whereas somatostatin induced a potent hyperpolarizing response. These studies demonstrate a relatively straightforward approach for transcriptome profiling of specific neuronal phenotypes. The stimulatory actions of GnRH and galanin upon GnRH neurons found here indicate that positive ultrashort feedback loops exist among the GnRH neuronal population.

  17. Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays

    PubMed Central

    Menzel, Ralph; Swain, Suresh C; Hoess, Sebastian; Claus, Evelyn; Menzel, Stefanie; Steinberg, Christian EW; Reifferscheid, Georg; Stürzenbaum, Stephen R

    2009-01-01

    Background Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. Results In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. Conclusion This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments. PMID:19366437

  18. Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts

    PubMed Central

    Trost, Brett; Moir, Catherine A.; Gillespie, Zoe E.; Kusalik, Anthony; Mitchell, Jennifer A.; Eskiw, Christopher H.

    2015-01-01

    DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data. PMID:26473061

  19. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.

    PubMed

    Fiegler, Heike; Carr, Philippa; Douglas, Eleanor J; Burford, Deborah C; Hunt, Sarah; Scott, Carol E; Smith, James; Vetrie, David; Gorman, Patricia; Tomlinson, Ian P M; Carter, Nigel P

    2003-04-01

    We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet.

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

    NASA Astrophysics Data System (ADS)

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

    2001-10-01

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

  1. Probe classification of on-off type DNA microarray images with a nonlinear matching measure

    NASA Astrophysics Data System (ADS)

    Ryu, Munho; Kim, Jong Dae; Min, Byoung Goo; Kim, Jongwon; Kim, Y. Y.

    2006-01-01

    We propose a nonlinear matching measure, called counting measure, as a signal detection measure that is defined as the number of on pixels in the spot area. It is applied to classify probes for an on-off type DNA microarray, where each probe spot is classified as hybridized or not. The counting measure also incorporates the maximum response search method, where the expected signal is obtained by taking the maximum among the measured responses of the various positions and sizes of the spot template. The counting measure was compared to existing signal detection measures such as the normalized covariance and the median for 2390 patient samples tested on the human papillomavirus (HPV) DNA chip. The counting measure performed the best regardless of whether or not the maximum response search method was used. The experimental results showed that the counting measure combined with the positional search was the most preferable.

  2. Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray.

    PubMed

    Bras, M; Dugas, V; Bessueille, F; Cloarec, J P; Martin, J R; Cabrera, M; Chauvet, J P; Souteyrand, E; Garrigues, M

    2004-11-01

    This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue.

  3. Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

    PubMed

    Gomes, Luciana I; Silva, Ricardo L A; Stolf, Beatriz S; Cristo, Elier B; Hirata, Roberto; Soares, Fernando A; Reis, Luiz F L; Neves, E Jordão; Carvalho, Alex F

    2003-10-15

    Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

  4. Capturing genomic signatures of DNA sequence variation using a standard anonymous microarray platform

    PubMed Central

    Cannon, C. H.; Kua, C. S.; Lobenhofer, E. K.; Hurban, P.

    2006-01-01

    Comparative genomics, using the model organism approach, has provided powerful insights into the structure and evolution of whole genomes. Unfortunately, only a small fraction of Earth's biodiversity will have its genome sequenced in the foreseeable future. Most wild organisms have radically different life histories and evolutionary genomics than current model systems. A novel technique is needed to expand comparative genomics to a wider range of organisms. Here, we describe a novel approach using an anonymous DNA microarray platform that gathers genomic samples of sequence variation from any organism. Oligonucleotide probe sequences placed on a custom 44 K array were 25 bp long and designed using a simple set of criteria to maximize their complexity and dispersion in sequence probability space. Using whole genomic samples from three known genomes (mouse, rat and human) and one unknown (Gonystylus bancanus), we demonstrate and validate its power, reliability, transitivity and sensitivity. Using two separate statistical analyses, a large numbers of genomic ‘indicator’ probes were discovered. The construction of a genomic signature database based upon this technique would allow virtual comparisons and simple queries could generate optimal subsets of markers to be used in large-scale assays, using simple downstream techniques. Biologists from a wide range of fields, studying almost any organism, could efficiently perform genomic comparisons, at potentially any phylogenetic level after performing a small number of standardized DNA microarray hybridizations. Possibilities for refining and expanding the approach are discussed. PMID:17000641

  5. Prospects for exploring the molecular-genomic foundations of therapeutic hypnosis with DNA microarrays.

    PubMed

    Rossi, Ernest Lawrence

    A new perspective on how therapeutic hypnosis and neuroscience may be integrated on the molecular-genomic level is offered as a guide for basic research and clinical applications. An update of Watson and Crick's original formulation of molecular biology is proposed to illustrate how psychosocial experiences modulate gene expression, protein synthesis, and brain plasticity during memory trace reactivation for the reorganization of neural networks that encode fear, stress, and traumatic symptoms. Examples of the scientific literature on DNA microarrays are used to explore how this new technology could integrate therapeutic hypnosis, neuroscience, and psychosocial genomics as a new foundation for mind-body medicine. Researchers and clinicians in therapeutic hypnosis need to partner with colleagues in neuroscience and molecular biology that utilize DNA microarray technology. It is recommended that hypnotic susceptibility scales of the future incorporate gene expression data to include the concept of "embodied imagination" and the "ideo-plastic faculty" on a molecular-genomic level as well as the psychological and behavioral level of ideomotor and ideosensory responses that are currently assessed.

  6. PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

    NASA Technical Reports Server (NTRS)

    Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

    2011-01-01

    Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated

  7. Long noncoding RNA expression profile of infantile hemangioma identified by microarray analysis.

    PubMed

    Liu, Xiaowen; Lv, Renrong; Zhang, Linfeng; Xu, Guangqi; Bi, Jianhai; Gao, Feng; Zhang, Jian; Xue, Feng; Wang, Fagang; Wu, Yiliang; Fu, Cong; Wang, Qiang; Huo, Ran

    2016-10-05

    Infantile hemangioma (IH) is one of the most common vascular tumors of childhood. Long noncoding RNAs (lncRNAs) play a critical role in angiogenesis, but their involvement in hemangioma remains unknown. This study aimed to assess the expression profiles of lncRNAs in IH and adjacent normal tissue samples, exploring the biological functions of lncRNAs as well as their involvement in IH pathogenesis. The lncRNA expression profiles were determined by lncRNA microarrays. A total of 1259 and 857 lncRNAs were upregulated and downregulated in IH, respectively, at a fold change cutoff of 2.0 (p < 0.05); in addition, 1469 and 1184 messenger RNAs (mRNAs) were upregulated and downregulated, respectively (fold change cutoff of 2.0; p < 0.05). A total of 292 differentially expressed mRNAs were targeted by the lncRNAs with altered expression in hemangioma, including 228 and 64 upregulated and downregulated, respectively (cutoff of 2.0, p < 0.05). Gene ontology (GO) analyses revealed several angiogenesis-related pathways. An lncRNA-mRNA co-expression network for differentially expressed lncRNAs revealed significant associations of the lncRNAs MEG3, MEG8, FENDRR, and Linc00152 with their related mRNAs. The validation results of nine differentially expressed lncRNAs (MALAT1, MEG3, MEG8, p29066, p33867, FENDRR, Linc00152, p44557_v4, p8683) as well as two mRNAs (FOXF1, EGFL7) indicated that the microarray data correlated well with the QPCR results. Interestingly, MALAT1 knockdown induced apoptosis and S-phase cell cycle arrest in human umbilical vein endothelial cells (HUVECs). Overall, this study revealed the lncRNA expression profile of IH and that lncRNAs likely regulate several genes with important roles in angiogenesis.

  8. Autoantigen Microarray for High-throughput Autoantibody Profiling in Systemic Lupus Erythematosus.

    PubMed

    Zhu, Honglin; Luo, Hui; Yan, Mei; Zuo, Xiaoxia; Li, Quan-Zhen

    2015-08-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components (nucleic acids and associated proteins), cytoplasmic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epitopes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection. Proteomic microarray as a multiplexed high-throughput screening platform is playing an increasingly-important role in autoantibody diagnostics. In this article, we highlight the use of autoantigen microarrays for autoantibody exploration in SLE.

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

    PubMed

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  10. Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

    SciTech Connect

    Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.; Speed, Terence P.; Rubin, Edward M.

    2000-05-05

    Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.

  11. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA).

    PubMed

    Rauch, Tibor A; Pfeifer, Gerd P

    2010-11-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

  12. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA)

    PubMed Central

    Rauch, Tibor A.; Pfeifer, Gerd P.

    2010-01-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences. PMID:20304072

  13. Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

    PubMed

    Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

    2013-04-01

    The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

  14. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays.

    PubMed

    Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

    2012-01-01

    PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.

  15. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

    PubMed Central

    Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

    2012-01-01

    PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

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

    NASA Astrophysics Data System (ADS)

    Kikuchi, Shoshi

    2009-02-01

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

  17. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice.

    PubMed

    Weindruch, R; Kayo, T; Lee, C K; Prolla, T A

    2001-03-01

    An active research area in biological gerontology concerns the mechanisms by which caloric restriction (CR) retards the aging process in laboratory rodents. We used high density oligonucleotide arrays representing 6347 genes to determine the gene expression profile of the aging process in gastrocnemius muscle of male C57BL/6 mice. Aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were completely or partially prevented by CR. Transcriptional patterns of muscle from calorie-restricted animals suggest that CR retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. The use of high density oligonucleotide microarrays provides a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of nutritional interventions designed to retard the aging process.

  18. Systematic Spatial Bias in DNA Microarray Hybridization Is Caused by Probe Spot Position-Dependent Variability in Lateral Diffusion

    PubMed Central

    Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

    2011-01-01

    Background The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. Methodology/Principal Findings This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Conclusions Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. PMID:21858215

  19. DNA nanofilm thickness measurement on microarray in air and in liquid using an atomic force microscope.

    PubMed

    Legay, Guillaume; Finot, Eric; Meunier-Prest, Rita; Cherkaoui-Malki, Mustapha; Latruffe, Norbert; Dereux, Alain

    2005-10-15

    The measurement of the thickness of DNA films on microarray as a function of the medium (liquid, air) is gaining importance for understanding the signal response of biosensors. Thiol group has been used to attach DNA strands to gold micropads deposited on silicon surface. Atomic force microscopy (AFM) was employed in its height mode to measure the change in the pad thickness and in its force mode to measure the indentation depth of the nanofilm. A good coherence between the height and force modes is observed for the film thickness in air. The adhesion force was found to be an alternative way to measure the surface coverage of the biolayer at nanoscopic scale. However the force analysis (compression, steric and electrostatic) provides baseline information necessary to interpret the AFM height image in liquid. Analysis of the film thickness distribution shows that the height of the DNA strands depends on both the DNA strand length (15-35 base pairs) and the environment (air, liquid). In air, longer strands lay down onto gold surface whereas the charge reversal of gold in liquid causes a repulsion of longer strands, which stand up.

  20. Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

    PubMed Central

    Widschwendter, Martin; Sun, Dahui; Sieburg, Hans B.; Spruck, Charles

    2010-01-01

    Background Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens. Methodology/Principal Findings In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay. Conclusions/Significance This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research. PMID:20596523

  1. Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

    PubMed

    Chen, Jie; Fu, Ziyi; Ji, Chenbo; Gu, Pingqing; Xu, Pengfei; Yu, Ningzhu; Kan, Yansheng; Wu, Xiaowei; Shen, Rong; Shen, Yan

    2015-05-01

    The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma.

  2. Challenges of microarray applications for microbial detection and gene expression profiling in food

    USDA-ARS?s Scientific Manuscript database

    Microarray technology represents one of the latest advances in molecular biology. The diverse types of microarrays have been applied to clinical and environmental microbiology, microbial ecology, and in human, veterinary, and plant diagnostics. Since multiple genes can be analyzed simultaneously, ...

  3. MELANCHOLIC DEPRESSION PREDICTION BY IDENTIFYING REPRESENTATIVE FEATURES IN METABOLIC AND MICROARRAY PROFILES WITH MISSING VALUES

    PubMed Central

    Nie, Zhi; Yang, Tao; Liu, Yashu; Lin, Binbin; Li, Qingyang; Narayan, Vaibhav A; Wittenberg, Gayle; Ye, Jieping

    2014-01-01

    Recent studies have revealed that melancholic depression, one major subtype of depression, is closely associated with the concentration of some metabolites and biological functions of certain genes and pathways. Meanwhile, recent advances in biotechnologies have allowed us to collect a large amount of genomic data, e.g., metabolites and microarray gene expression. With such a huge amount of information available, one approach that can give us new insights into the understanding of the fundamental biology underlying melancholic depression is to build disease status prediction models using classification or regression methods. However, the existence of strong empirical correlations, e.g., those exhibited by genes sharing the same biological pathway in microarray profiles, tremendously limits the performance of these methods. Furthermore, the occurrence of missing values which are ubiquitous in biomedical applications further complicates the problem. In this paper, we hypothesize that the problem of missing values might in some way benefit from the correlation between the variables and propose a method to learn a compressed set of representative features through an adapted version of sparse coding which is capable of identifying correlated variables and addressing the issue of missing values simultaneously. An efficient algorithm is also developed to solve the proposed formulation. We apply the proposed method on metabolic and microarray profiles collected from a group of subjects consisting of both patients with melancholic depression and healthy controls. Results show that the proposed method can not only produce meaningful clusters of variables but also generate a set of representative features that achieve superior classification performance over those generated by traditional clustering and data imputation techniques. In particular, on both datasets, we found that in comparison with the competing algorithms, the representative features learned by the proposed

  4. Melancholic depression prediction by identifying representative features in metabolic and microarray profiles with missing values.

    PubMed

    Nie, Zhi; Yang, Tao; Liu, Yashu; Li, Qingyang; Narayan, Vaibhav A; Wittenberg, Gayle; Ye, Jieping

    2015-01-01

    Recent studies have revealed that melancholic depression, one major subtype of depression, is closely associated with the concentration of some metabolites and biological functions of certain genes and pathways. Meanwhile, recent advances in biotechnologies have allowed us to collect a large amount of genomic data, e.g., metabolites and microarray gene expression. With such a huge amount of information available, one approach that can give us new insights into the understanding of the fundamental biology underlying melancholic depression is to build disease status prediction models using classification or regression methods. However, the existence of strong empirical correlations, e.g., those exhibited by genes sharing the same biological pathway in microarray profiles, tremendously limits the performance of these methods. Furthermore, the occurrence of missing values which are ubiquitous in biomedical applications further complicates the problem. In this paper, we hypothesize that the problem of missing values might in some way benefit from the correlation between the variables and propose a method to learn a compressed set of representative features through an adapted version of sparse coding which is capable of identifying correlated variables and addressing the issue of missing values simultaneously. An efficient algorithm is also developed to solve the proposed formulation. We apply the proposed method on metabolic and microarray profiles collected from a group of subjects consisting of both patients with melancholic depression and healthy controls. Results show that the proposed method can not only produce meaningful clusters of variables but also generate a set of representative features that achieve superior classification performance over those generated by traditional clustering and data imputation techniques. In particular, on both datasets, we found that in comparison with the competing algorithms, the representative features learned by the proposed

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

    SciTech Connect

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

    2004-12-17

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

  6. Enzymatic on-chip enhancement for high resolution genotyping DNA microarrays.

    PubMed

    Schulze, Holger; Barl, Timo; Vase, Hollie; Baier, Shiromi; Thomas, Peter; Giraud, Gerard; Crain, Jason; Bachmann, Till T

    2012-06-05

    Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant Klebsiella pneumoniae carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination.

  7. A comparison of parametric and nonparametric methods for normalising cDNA microarray data.

    PubMed

    Khondoker, Mizanur R; Glasbey, Chris A; Worton, Bruce J

    2007-12-01

    Normalisation is an essential first step in the analysis of most cDNA microarray data, to correct for effects arising from imperfections in the technology. Loess smoothing is commonly used to correct for trends in log-ratio data. However, parametric models, such as the additive plus multiplicative variance model, have been preferred for scale normalisation, though the variance structure of microarray data may be of a more complex nature than can be accommodated by a parametric model. We propose a new nonparametric approach that incorporates location and scale normalisation simultaneously using a Generalised Additive Model for Location, Scale and Shape (GAMLSS, Rigby and Stasinopoulos, 2005, Applied Statistics, 54, 507-554). We compare its performance in inferring differential expression with Huber et al.'s (2002, Bioinformatics, 18, 96-104) arsinh variance stabilising transformation (AVST) using real and simulated data. We show GAMLSS to be as powerful as AVST when the parametric model is correct, and more powerful when the model is wrong. (c) 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  8. DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

    PubMed

    Feroudj, Holger; Matsumoto, Takuya; Kurosu, Yohei; Kaneko, Gen; Ushio, Hideki; Suzuki, Katsuaki; Kondo, Hidehiro; Hirono, Ikuo; Nagashima, Yuji; Akimoto, Seiji; Usui, Kazushige; Kinoshita, Shigeharu; Asakawa, Shuichi; Kodama, Masaaki; Watabe, Shugo

    2014-01-01

    Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

    PubMed

    Quintela, T; Marcelino, H; Deery, M J; Feret, R; Howard, J; Lilley, K S; Albuquerque, T; Gonçalves, I; Duarte, A C; Santos, C R A

    2016-01-01

    The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

  10. Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

    PubMed

    Renault, N K; Gaddipati, S R; Wulfert, F; Falcone, F H; Mirotti, L; Tighe, P J; Wright, V; Alcocer, M J C

    2011-02-01

    Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described

  11. Probing plant-pathogen interactions and downstream defense signaling using DNA microarrays.

    PubMed

    Wan, Jinrong; Dunning, F Mark; Bent, Andrew F

    2002-11-01

    The interaction between a plant and a pathogen activates a wide variety of defense responses. The recent development of microarray-based expression profiling methods, together with the availability of genomic and/or EST (expressed sequence tag) sequence data for some plant species, has allowed significant progress in the characterization of plant pathogenesis-related responses. The small number of expression profiling studies completed to date have already identified an amazing number of genes that had not previously been implicated in plant defense. Some of these genes can be associated with defense signal transduction or antimicrobial action, but the functional contribution of many others remains uncertain. Initial expression profiling work has also revealed similarities and distinctions between different defense signaling pathways, and cross-talk (both overlap and interference) between pathogenesis-related responses and plant responses to other stresses. Potential transcriptional cis-regulatory elements upstream of co-regulated genes can also be identified. Whole-genome arrays are only now becoming available, and many interactions remain to be studied (e.g. different pathogen species, plant genotypes, mutants, time-points after infection). Expression profiling technologies, in combination with other genomic tools, will have a substantial impact on our understanding of plant-pathogen interactions and defense signaling pathways.

  12. ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

    USDA-ARS?s Scientific Manuscript database

    We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

  13. Progression in cutaneous malignant melanoma is associated with distinct expression profiles: a tissue microarray-based study.

    PubMed

    Alonso, Soledad R; Ortiz, Pablo; Pollán, Marina; Pérez-Gómez, Beatriz; Sánchez, Lydia; Acuña, Ma Jesús; Pajares, Raquel; Martínez-Tello, Francisco J; Hortelano, Carlos M; Piris, Miguel A; Rodríguez-Peralto, José L

    2004-01-01

    Cutaneous malignant melanoma remains the leading cause of skin cancer death in industrialized countries. Clinical and histological variables that predict survival, such as Breslow's index, tumor size, ulceration, or vascular invasion have been identified in malignant melanoma. Nevertheless, the potential relevance of biological variables still awaits an in-depth exploration. Using tissue microarrays (TMAs), we retrospectively analyzed 165 malignant melanoma samples from 88 patients corresponding to distinct histological progression phases, radial, vertical, and metastases. A panel of 39 different antibodies for cell cycle, apoptosis, melanoma antigens, transcription factors, DNA mismatch repair, and other proteins was used. Integrating the information, the study has identified expression profiles distinguishing specific melanoma progression stages. Most of the detected alterations were linked to the control of cell cycle G1/S transition; cyclin D1 was expressed in radial cases 48% (12 of 25) with significant lost of expression in vertical cases 14% (9 of 65), P = 0.002; whereas p16(INK4a) (89% in vertical versus 71% in metastatic cases, P = 0.009) and p27(KIP1) (76% in radial versus 45% in vertical cases, P = 0.010) were diminished in advanced stages. The study also defines a combination of biological markers associated with shorter overall survival in patients with vertical growth phase melanoma, that provided a predictor model with four antibodies (Ki67, p16(INK4a), p21(CIP1), and Bcl-6). This predictor model was validated using an independent series of 72 vertical growth phase melanoma patients.

  14. Administered chrysanthemum flower oil attenuates hyperuricemia: mechanism of action as revealed by DNA microarray analysis.

    PubMed

    Honda, Shinichi; Kawamoto, Seiji; Tanaka, Hozumi; Kishida, Hideyuki; Kitagawa, Masayasu; Nakai, Yuji; Abe, Keiko; Hirata, Dai

    2014-01-01

    We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.

  15. Convergent evolution to an aptamer observed in small populations on DNA microarrays

    NASA Astrophysics Data System (ADS)

    Rowe, W.; Platt, M.; Wedge, D. C.; Day, P. J. R.; Kell, D. B.; Knowles, J. D.

    2010-09-01

    The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.

  16. Fluorescence lifetime biosensing with DNA microarrays and a CMOS-SPAD imager

    PubMed Central

    Giraud, Gerard; Schulze, Holger; Li, Day-Uei; Bachmann, Till T.; Crain, Jason; Tyndall, David; Richardson, Justin; Walker, Richard; Stoppa, David; Charbon, Edoardo; Henderson, Robert; Arlt, Jochen

    2010-01-01

    Fluorescence lifetime of dye molecules is a sensitive reporter on local microenvironment which is generally independent of fluorophores concentration and can be used as a means of discrimination between molecules with spectrally overlapping emission. It is therefore a potentially powerful multiplexed detection modality in biosensing but requires extremely low light level operation typical of biological analyte concentrations, long data acquisition periods and on-chip processing capability to realize these advantages. We report here fluorescence lifetime data obtained using a CMOS-SPAD imager in conjunction with DNA microarrays and TIRF excitation geometry. This enables acquisition of single photon arrival time histograms for a 320 pixel FLIM map within less than 26 seconds exposure time. From this, we resolve distinct lifetime signatures corresponding to dye-labelled HCV and quantum-dot-labelled HCMV nucleic acid targets at concentrations as low as 10 nM. PMID:21258550

  17. Combining suppressive subtractive hybridization and cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney.

    PubMed

    Lake, Jennifer; Gravel, Catherine; Koko, Gabriel Koffi D; Robert, Claude; Vandenberg, Grant W

    2010-03-01

    Phosphorus (P)-responsive genes and how they regulate renal adaptation to phosphorous-deficient diets in animals, including fish, are not well understood. RNA abundance profiling using cDNA microarrays is an efficient approach to study nutrient-gene interactions and identify these dietary P-responsive genes. To test the hypothesis that dietary P-responsive genes are differentially expressed in fish fed varying P levels, rainbow trout were fed a practical high-P diet (R20: 0.96% P) or a low-P diet (R0: 0.38% P) for 7 weeks. The differentially-expressed genes between dietary groups were identified and compared from the kidney by combining suppressive subtractive hybridization (SSH) with cDNA microarray analysis. A number of genes were confirmed by real-time PCR, and correlated with plasma and bone P concentrations. Approximately 54 genes were identified as potential dietary P-responsive after 7 weeks on a diet deficient in P according to cDNA microarray analysis. Of 18 selected genes, 13 genes were confirmed to be P-responsive at 7 weeks by real-time PCR analysis, including: iNOS, cytochrome b, cytochrome c oxidase subunit II , alpha-globin I, beta-globin, ATP synthase, hyperosmotic protein 21, COL1A3, Nkef, NDPK, glucose phosphate isomerase 1, Na+/H+ exchange protein and GDP dissociation inhibitor 2. Many of these dietary P-responsive genes responded in a moderate way (R0/R20 ratio: <2-3 or >0.5) and in a transient manner to dietary P limitation. In summary, renal adaptation to dietary P deficiency in trout involves changes in the expression of several genes, suggesting a profile of metabolic stress, since many of these differentially-expressed candidates are associated with the cellular adaptative responses.

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

    PubMed Central

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

    2015-01-01

    AIM: To investigate the impact of MK-801 on gene expression patterns genome wide in rat brain regions. METHODS: Rats were treated with an intraperitoneal injection of MK-801 [0.08 (low-dose) and 0.16 (high-dose) mg/kg] or NaCl (vehicle control). In a first series of experiment, the frontoparietal electrocorticogram was recorded 15 min before and 60 min after injection. In a second series of experiments, the whole brain of each animal was rapidly removed at 40 min post-injection, and different regions were separated: amygdala, cerebral cortex, hippocampus, hypothalamus, midbrain and ventral striatum on ice followed by DNA microarray (4 × 44 K whole rat genome chip) analysis. RESULTS: Spectral analysis revealed that a single systemic injection of MK-801 significantly and selectively augmented the power of baseline gamma frequency (30-80 Hz) oscillations in the frontoparietal electroencephalogram. DNA microarray analysis showed the largest number (up- and down- regulations) of gene expressions in the cerebral cortex (378), midbrain (376), hippocampus (375), ventral striatum (353), amygdala (301), and hypothalamus (201) under low-dose (0.08 mg/kg) of MK-801. Under high-dose (0.16 mg/kg), ventral striatum (811) showed the largest number of gene expression changes. Gene expression changes were functionally categorized to reveal expression of genes and function varies with each brain region. CONCLUSION: Acute MK-801 treatment increases synchrony of baseline gamma oscillations, and causes very early changes in gene expressions in six individual rat brain regions, a first report. PMID:26629322

  19. Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

    PubMed Central

    La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

    2009-01-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

  20. Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

    PubMed

    La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

    2009-10-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

  1. Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

    PubMed

    Chiu, Sung-Kay; Hsu, Mandy; Ku, Wei-Chi; Tu, Ching-Yu; Tseng, Yu-Tien; Lau, Wai-Kwan; Yan, Rong-Yih; Ma, Jing-Tyan; Tzeng, Chi-Meng

    2003-09-15

    Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

  2. Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

    PubMed Central

    Diehl, Frank; Grahlmann, Susanne; Beier, Markus; Hoheisel, Jörg D.

    2001-01-01

    Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface. PMID:11266573

  3. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  4. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  5. Oligonucleotide Microarray Analysis of Dietary-Induced Hyperlipidemia Gene Expression Profiles in Miniature Pigs

    PubMed Central

    Takahashi, Junko; Waki, Shiori; Matsumoto, Rena; Odake, Junji; Miyaji, Takayuki; Tottori, Junichi; Iwanaga, Takehiro; Iwahashi, Hitoshi

    2012-01-01

    Background Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. Methodology Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF) Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD) and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD). Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. Principal Findings Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO) based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. Conclusions No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in dietary

  6. Automatic image analysis and spot classification for detection of pathogenic Escherichia coli on glass slide DNA microarrays

    USDA-ARS?s Scientific Manuscript database

    A computer algorithm was created to inspect scanned images from DNA microarray slides developed to rapidly detect and genotype E. Coli O157 virulent strains. The algorithm computes centroid locations for signal and background pixels in RGB space and defines a plane perpendicular to the line connect...

  7. An undergraduate laboratory exercise to study the effect of darkness on plant gene expression using DNA microarray.

    PubMed

    Chang, Ming-Mei; Briggs, George M

    2007-11-01

    DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory exercise using an Arabidopsis DNA microarray to study the gene expression of Brassica rapa, Wisconsin Fast Plant. Genes involved in senescence, cell wall loosening/degradation, and sugar transport were the most upregulated, while those involved in photosynthesis, the elimination of reactive oxygen intermediates associated with photooxidative stress and auxin synthesis, were the most downregulated. Students were able to complete the experiment successfully. Throughout the exercise, they learned various important molecular techniques including RNA isolation, quantification, reverse transcription, cRNA synthesis, labeling and purification, and microarray hybridization, washing, scanning, and feature extraction. The exercise can be integrated into a college-level molecular biology laboratory. The procedure used can be adapted to examine other effects on other organisms. Copyright © 2007 International Union of Biochemistry and Molecular Biology, Inc.

  8. Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.

    PubMed

    Marokhazi, Judit; Waterfield, Nicholas; LeGoff, Gaelle; Feil, Edward; Stabler, Richard; Hinds, Jason; Fodor, Andras; ffrench-Constant, Richard H

    2003-08-01

    Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.

  9. Using a DNA Microarray To Investigate the Distribution of Insect Virulence Factors in Strains of Photorhab