Long synthetic oligonucleotides for microarray expression measurement
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There are generally two kinds of DNA microarray used for genomic-scale gene expression profiling of mRNA: cDNA and DNA chip, but both of them suffer from some drawbacks. To meet more requirements, another oligonucleotide microarray with long was produced. This type of microarray had the advantages of low cost, minimal Cross-hybridization, flexible and easy to make, which is most fit for small laboratories with special purposes. In this paper, we devised different probes with different probe lengths, GC contents and gene positions to optimization the probe design. Experiments showed 70 mer probes are suitable for both sufficient sensitivity and reasonable costs. Higher G-C content produces stronger signal intensity thus better sensitivity and probes designed at 3 untranslated region of gene within the range of 300 pb should be best for both sensitivity and specificity.Keywords:
Gene chip analysis
At present, there are no clinically reliable disease markers for chronic fatigue syndrome. DNA chip microarray technology provides a method for examining the differential expression of mRNA from a large number of genes. Our hypothesis was that a gene expression signature, generated by microarray assays, could help identify genes which are dysregulated in patients with post-infectious CFS and so help identify biomarkers for the condition.Human genome-wide Affymetrix GeneChip arrays (39,000 transcripts derived from 33,000 gene sequences) were used to compare the levels of gene expression in the peripheral blood mononuclear cells of male patients with post-infectious chronic fatigue (n = 8) and male healthy control subjects (n = 7).Patients and healthy subjects differed significantly in the level of expression of 366 genes. Analysis of the differentially expressed genes indicated functional implications in immune modulation, oxidative stress and apoptosis. Prototype biomarkers were identified on the basis of differential levels of gene expression and possible biological significanceDifferential expression of key genes identified in this study offer an insight into the possible mechanism of chronic fatigue following infection. The representative biomarkers identified in this research appear promising as potential biomarkers for diagnosis and treatment.
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Gene chip analysis
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Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes.Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 degrees C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 degrees C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations.This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.
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cDNA microarrays are powerful tools to analyze the expression pattern of thousands of genes simultaneously in response to treatments, during tissue differentiation or related to differences in traits. To get accurate results, the probes used in the microarray are to be specific to the species for which the array is being conducted. Non-availability of suitable microarray specific to mithun (Bos frontalis), a rare ruminant of Southeast Asia, limits its use to study the expression patterns of genes globally. Therefore, the authors designed the probes and developed a mithun-specific microarray for the first time. Transcripts obtained from bovine genome array in mithun tissues and from BLAST search were used to design probes and subsequent development of mithun-specific microarray. A total of 3,35,576 numbers of transcripts obtained from the next generation sequencing (through microarray using bovine genome array chips) of mithun tissues including several hundreds of unknown genes and others obtained for BLAST search (11,865) were used for designing the probes towards development of mithun-specific microarrays. A total of 3,47,441 probes were designed and length of each probe was 60 bp. The developed array has total features of 4,20,288, of which 4,16,505 features are readily available in the developed array. Besides, there are 3783 numbers of controls in the array to obtain high performance. In conclusion, the authors developed mithun-specific microarray using probes that are specific to this species for the first time. This microarray for mithun contains fully defined set of genes suitable for detailed analysis of expression of many genes involved in this species. The developed microarray will be of great use to analyze the molecular mechanisms controlling cell proliferation, differentiation in response to different treatments or variation in economic traits such as marbling and disease resistance in mithun. Keywords: Mithun, microarray, cDNA, transcripts, expression, genes Cite this Article Mondal M, Karunakaran M, Baruah KK. Designing mithun specific probes towards development of microarray for mithun (Bos frontalis). Research & Reviews: Journal of Veterinary Science and Technology. 2015; 4(3): 1–7p.
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The introduction of microarray technology has dramatically changed the way that researchers address many biomedical questions. DNA microarrays can measure expression of thousands of genes simultaneously, providing extensive information on gene interaction and function. Microarray technology is a powerful tool for identifying novel molecular drug targets and for elucidating mechanisms of drug action. Furthermore, microarrays can monitor the global profile of gene expression in response to specific pharmacologic agents, providing information on drug efficacy and toxicity. Over the last several years, dramatic advancements have occurred in array technology. In this review we describe basic aspects of microarray instrumentation and experimentation. Each of the major array formats including oligonucleotides arrays, spotted arrays, and macroarrays are examined, and advantages and options for using each format are presented. Important factors in the design and analysis of microarray experiments are also discussed. Most importantly, we explore recent developments in microarray technology that are relevant to pharmacogenomics and the discovery of gene function.
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DNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially. In this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene. In view of these pitfalls, data from microarray analysis need to be interpreted cautiously.
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Although microarray analysis is a highly promising technology in the genome era, its application for gene expression profiling to characterize various phenomes, including genetic phenotypes, diseases, responses to chemicals and clinical annotations, is far from being a real use. One of the obstacles is the quality of the data, which needs to be enough to be able to solely use microarrays for these purposes. For this, selecting a set of genes as a molecular signature, based on transcriptomics, proteomics or metabolomics, and the use of the selected set of genes in focused microarrays has great merits. Here, we summarize how sets of genes were selected, what types of genes were used and what kind of statistics will be needed for focused microarrays, to distinguish them from genome-wide microarrays and to explain why focused microarray analysis is advantageous in gene expression profiling. Keywords: cDNA array, genome-wide microarray, Replicate Assays, EUROSTERONE microarray, Signaling Pathways
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High‐throughput microarray technologies have the potential to impact significantly on the practice of histopathology over the coming years. Global gene expression profiling allows for a systematic search of all human genes for novel diagnostic and prognostic markers and for potential therapeutic targets. Likewise, gene copy number changes can be determined on a gene‐by‐gene basis using microarrays. Tissue microarrays are an efficient method to extend and validate the findings obtained from the initial ‘discovery’ phase of the research, done using cDNA microarrays. In addition, tissue microarrays can be used for quality assurance for immunohistochemical and in situ hybridization procedures. In this review we give a brief overview of microarray technology and research uses, and discuss potential applications of microarrays in the practice of diagnostic histopathology.
Histopathology
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