Quantification of global transcription patterns in prokaryotes using spotted microarrays.
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ABSTRACT:
SUBMITTER: Sidders B
PROVIDER: S-EPMC2246267 | biostudies-literature | 2007
REPOSITORIES: biostudies-literature
Publications
Quantification of global transcription patterns in prokaryotes using spotted microarrays.
Sidders Ben B Withers Mike M Kendall Sharon L SL Bacon Joanna J Waddell Simon J SJ Hinds Jason J Golby Paul P Movahedzadeh Farahnaz F Cox Robert A RA Frita Rosangela R Ten Bokum Annemieke M C AM Wernisch Lorenz L Stoker Neil G NG
Genome biology 20070101 12
We describe an analysis, applicable to any spotted microarray dataset produced using genomic DNA as a reference, that quantifies prokaryotic levels of mRNA on a genome-wide scale. Applying this to Mycobacterium tuberculosis, we validate the technique, show a correlation between level of expression and biological importance, define the complement of invariant genes and analyze absolute levels of expression by functional class to develop ways of understanding an organism's biology without comparis ...[more]
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Project description:We describe an analysis, applicable to any spotted microarray dataset produced using genomic DNA as a reference, which quantifies prokaryotic levels of mRNA on a genome-wide scale. Applying this to Mycobacterium tuberculosis, we validate the technique, show a correlation between level of expression and biological importance, define the complement of invariant genes and analyse absolute levels of expression by functional class to develop ways of understanding an organism's biology without comparison to another growth condition. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-60
2008-09-30 | E-BUGS-60 | biostudies-arrayexpress
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Project description:BackgroundMicroarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on three Gossypium species. Using these assemblies as templates, we describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species.ResultsSynthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included on the arrays, optimized to target the diversity of the transcriptome and previously studied cotton genes, transcription factors, and genes with homology to Arabidopsis. A small portion of the oligonucleotides target unidentified protein coding sequences, thereby providing an element of gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA libraries, the microarray has direct application for analysis of the fiber transcriptome. To illustrate the utility of the microarray, we hybridized labeled bud and leaf cDNAs from G. hirsutum and demonstrate technical consistency of results.ConclusionThe cotton oligonucleotide microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info.
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Project description:The transport of carbohydrates by Streptococcus mutans is accomplished by the phosphoenolpyruvate-phosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters. To undertake a global transcriptional analysis of all S. mutans sugar transporters simultaneously, we used a whole-genome expression microarray. Global transcription profiles of S. mutans UA159 were determined for several monosaccharides (glucose, fructose, galactose, and mannose), disaccharides (sucrose, lactose, maltose, and trehalose), a beta-glucoside (cellobiose), oligosaccharides (raffinose, stachyose, and maltotriose), and a sugar alcohol (mannitol). The results revealed that PTSs were responsible for transport of monosaccharides, disaccharides, beta-glucosides, and sugar alcohol. Six PTSs were transcribed only if a specific sugar was present in the growth medium; thus, they were regulated at the transcriptional level. These included transporters for fructose, lactose, cellobiose, and trehalose and two transporters for mannitol. Three PTSs were repressed under all conditions tested. Interestingly, five PTSs were always highly expressed regardless of the sugar source used, presumably suggesting their availability for immediate uptake of most common dietary sugars (glucose, fructose, maltose, and sucrose). The ABC transporters were found to be specific for oligosaccharides, raffinose, stachyose, and isomaltosaccharides. Compared to the PTSs, the ABC transporters showed higher transcription under several tested conditions, suggesting that they might be transporting multiple substrates.
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Project description:PurposeThe present study was designed to compare the results obtained from two different microarray platforms: spotted cDNAs using a two-color system (Clontech, Atlas Glass Human 3.8) and the Affymetrix platform. We evaluated the internal consistency within each of the platforms, and compared the results across the two platforms.MethodsRNA was isolated from two retinal pigment epithelial (RPE) cell lines, D407 cells and ARPE19 cells. Each microarray system requires a specific RNA isolation and target preparation procedure. To compare the results between the two platforms, the intensity values for each platform were standardized and scaled. This allowed for a direct comparison of the data between two very different microarray platforms. Real-time RT-PCR was used as an independent conformation of expression levels for selected transcripts. The protein levels for some of these genes were determined using a quantitative immunoblot method.ResultsFirst, we compared the transcriptome of the D407 cell line to itself. Within each of the platforms there was a high degree of consistency. However, when the data from the Atlas Glass Human 3.8 microarray platform was compared to that of the Affymetrix platform there was a dramatic lack of agreement. The second step was to compare the mRNA profile of the ARPE19 cell line to the D407 cell line. Again there was good agreement within each platform. When the results of the Atlas Glass Human 3.8 platform were compared to the Affymetrix platform, there was a surprising lack of agreement between the two data sets. Real-time RT-PCR was used as independent means of defining RNA levels in the two cell lines. In general, the real-time RT-PCR results were in better agreement with the Affymetrix platform (85%) than the Atlas Glass platform (33%). In addition, we also examined the levels of 11 proteins in these two cell lines using a quantitative immunoblot method. The results from this protein analysis had a higher degree of concordance with the results from Affymetrix platform.ConclusionsIn both the Atlas Glass Human 3.8 system and the Affymetrix platform, there is a high degree of internal consistency. However, comparisons between the two platforms show a lack of agreement. In general, the real-time RT-PCR confirmed the results on the Affymetrix system more often than those from Atlas Glass arrays. However, in both cases, conformation by an independent method proves to be of considerable value.
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Project description:Microarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, a global assembly of cotton ESTs was constructed based on three Gossypium. Using that assembly as a template, we now describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species. Synthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of more than 150,000 cotton ESTs derived from 30 different cDNA libraries representing many different tissue types and tissue treatments. A total of 13,158 oligonucleotide probes are included on the arrays, optimized to target the diversity of the transcriptome but also including previously studied cotton genes, duplicated gene pairs derived from a paleoduplication event, transcription factors, and homology to protein coding genes in Arabidopsis. About 10% of the oligonucleotides target unidentified protein coding sequences, thereby providing an element of gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA libraries, the array has direct application for analysis of the fiber transcriptome. To illustrate the utility of the array, we hybridized labeled bud and leaf cDNAs from G. hirsutum and demonstrate technical consistency of results. The cotton microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info. Keywords: self vs. self; platform testing
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Project description:BackgroundSpotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard.ResultsWe performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable.ConclusionRefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.
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Project description:BackgroundRNA-Seq exploits the rapid generation of gigabases of sequence data by Massively Parallel Nucleotide Sequencing, allowing for the mapping and digital quantification of whole transcriptomes. Whilst previous comparisons between RNA-Seq and microarrays have been performed at the level of gene expression, in this study we adopt a more fine-grained approach. Using RNA samples from a normal human breast epithelial cell line (MCF-10a) and a breast cancer cell line (MCF-7), we present a comprehensive comparison between RNA-Seq data generated on the Applied Biosystems SOLiD platform and data from Affymetrix Exon 1.0ST arrays. The use of Exon arrays makes it possible to assess the performance of RNA-Seq in two key areas: detection of expression at the granularity of individual exons, and discovery of transcription outside annotated loci.ResultsWe found a high degree of correspondence between the two platforms in terms of exon-level fold changes and detection. For example, over 80% of exons detected as expressed in RNA-Seq were also detected on the Exon array, and 91% of exons flagged as changing from Absent to Present on at least one platform had fold-changes in the same direction. The greatest detection correspondence was seen when the read count threshold at which to flag exons Absent in the SOLiD data was set to t<1 suggesting that the background error rate is extremely low in RNA-Seq. We also found RNA-Seq more sensitive to detecting differentially expressed exons than the Exon array, reflecting the wider dynamic range achievable on the SOLiD platform. In addition, we find significant evidence of novel protein coding regions outside known exons, 93% of which map to Exon array probesets, and are able to infer the presence of thousands of novel transcripts through the detection of previously unreported exon-exon junctions.ConclusionsBy focusing on exon-level expression, we present the most fine-grained comparison between RNA-Seq and microarrays to date. Overall, our study demonstrates that data from a SOLiD RNA-Seq experiment are sufficient to generate results comparable to those produced from Affymetrix Exon arrays, even using only a single replicate from each platform, and when presented with a large genome.
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Project description:The transport and catabolic machinery involved in carbohydrate utilization by Lactobacillus acidophilus was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a round-robin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phosphoenolpyruvate:sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactoside-pentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK/P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided. L. acidophilus's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.
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