Assessing the importance of target type for oligonucleotide microarray experiments
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ABSTRACT:
ORGANISM(S): Methylosinus trichosporium Dehalococcoides mccartyi VS Sphingomonas paucimobilis Methylosinus trichosporium OB3b
PROVIDER: GSE23168 | GEO | 2010/07/27
SECONDARY ACCESSION(S): PRJNA131559
REPOSITORIES: GEO
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Project description:In order to assess the effect on signal variability of the type of target used for oligonucleotide microarray experiments, we hybridized double and single stranded DNA of three genes on a Nimblegen High Density microarray. This slide carries specific probes (two lengths: 30 and 50 nucleotides, for a total of 7456 probes) targeting every region of selected genes. Probes of 30 nucleotides and 50 nucleotides, covering the full length of three genes (ahdA1C, mmoC and vcrA) by a one nucleotide step, were designed and in situ synthesized in six replicates on a 4*72K Nimblegen Custom Microarray. Double and single stranded DNA of the three genes were hybridized as target.
2010-07-27 | E-GEOD-23168 | biostudies-arrayexpress
Project description:Assessing the importance of target type for oligonucleotide microarray experiments
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2013-03-31 | GSE28208 | GEO
Project description:This study describes the development and validation of the Aquagenomic Sparus aurata oligonucleotide-microarray (SAQ) based on the Agilent Technology system (eArray) to provide a platform for studies on the gene expression of gilthead seabream. The platform developed used all available public ESTs stored and annotated in the Aquagenomic Consortium seabream library (10K). In fish, lipopolysaccharide (LPS) gives a robust cytokine response that is stimulated by crude LPS preparations, some component of the LPS complex are responsible for this stimulation. Peptidoglycan (PGN) is a component of G-negative bacteria (found as a contaminant of crude LPS preparations) able to be recognized by macrophages inducing depth transcriptional modulations and a strong inflammatory response. For microarray analysis, head kidney macrophage cultures were used (N = 36 fish). Each cell culture was stimulated with equal concentrations of PGN and LPS from E. coli O111:B4 strain (10 ug/mL): non-stimulated cell cultures (control n = 9 fish), stimulated during 6 h with LPS (n = 9), and stimulated during 1 h (n = 9), and 6 h (n = 9) with PGN. A loop microarray design approach was used for the study. All experimental RNA samples were labelled with a single colour dye (Cy3) and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Our microarray analyses identified differential transcriptional modulations in macrophages stimulated with both LPS and PGN at the level of differentially activated RNA transcripts related with the regulation of transcriptional program, prostaglandin synthesis or highlighting the expression of responsive gene-cassettes tightly related to LPS-PGN host recognition. A total of 43,398 oligonucleotide probes were used to construct a high-density seabream microarray based on the Agilent 4 × 44 K design format. Microarray hybridization validation was made by analyzing the gene expression profiles in primary cultures of seabream macrophages (MC). 7,285 transcripts with annotated sequences were spotted in triplicate onto the slide (total probes 21,855), as well as 8,377 ESTs without annotation, 183 enriched sequences (gene bank) with 15 replicated probes (total probes 2,745), and finally 1,417 internal control probes of Agilent (N = 43,398).
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Project description:To obtain the gene expression profiles of the moss Physcomitrella patens ABA insensitive mutant AR7, we performed microarray analysis of wildtype plant and AR7 using a custom Physcomitrella oligonucleotide microarray, which carries probes for 33,942 gene models of Physcomitrella genome version 1.1. on a 4 x 44 K Agilent platform.
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Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age.
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Project description:cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high.
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Project description:DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays
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