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Spotted cotton oligonucleotide microarrays for gene expression analysis.

ABSTRACT: BACKGROUND: Microarrays 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. RESULTS: Synthetic 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. CONCLUSION: The cotton oligonucleotide microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info.

SUBMITTER: Udall JA

PROVIDER: S-EPMC3225879 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Spotted cotton oligonucleotide microarrays for gene expression analysis.

Udall Joshua A JA Flagel Lex E LE Cheung Foo F Woodward Andrew W AW Hovav Ran R Rapp Ryan A RA Swanson Jordan M JM Lee Jinsuk J JJ Gingle Alan R AR Nettleton Dan D Town Christopher D CD Chen Z Jeffrey ZJ Wendel Jonathan F JF

BMC genomics 20070327

<h4>Background</h4>Microarrays 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.<h4>Results</h4>Synthetic oligonucleotide probes were generated from exemplar sequences of a global ...[more]

PMID: 17389046

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Project description:To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest.The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb.Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

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.

Project description:BACKGROUND: Gene expression profiles of non-model mammals may provide valuable data for biomedical and evolutionary studies. However, due to lack of sequence information of other species, DNA microarrays are currently restricted to humans and a few model species. This limitation may be overcome by using arrays developed for a given species to analyse gene expression in a related one, an approach known as "cross-species analysis". In spite of its potential usefulness, the accuracy and reproducibility of the gene expression measures obtained in this way are still open to doubt. The present study examines whether or not hybridization values from cross-species analyses are as reproducible as those from same-species analyses when using Affymetrix oligonucleotide microarrays. RESULTS: The reproducibility of the probe data obtained hybridizing deer, Old-World primates, and human RNA samples to Affymetrix human GeneChip U133 Plus 2.0 was compared. The results show that cross-species hybridization affected neither the distribution of the hybridization reproducibility among different categories, nor the reproducibility values of the individual probes. Our analyses also show that a 0.5% of the probes analysed in the U133 plus 2.0 GeneChip are significantly associated to un-reproducible hybridizations. Such probes-called in the text un-reproducible probe sequences- do not increase in number in cross-species analyses. CONCLUSION: Our study demonstrates that cross-species analyses do not significantly affect hybridization reproducibility of GeneChips, at least within the range of the mammal species analysed here. The differences in reproducibility between same-species and cross-species analyses observed in previous studies were probably caused by the analytical methods used to calculate the gene expression measures. Together with previous observations on the accuracy of GeneChips for cross-species analysis, our analyses demonstrate that cross-species hybridizations may provide useful gene expression data. However, the reproducibility and accuracy of these measures largely depends on the use of appropriated algorithms to derive the gene expression data from the probe data. Also, the identification of probes associated to un-reproducible hybridizations-useless for gene expression analyses- in the studied GeneChip, stress the need of a re-evaluation of the probes' performance.

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Spotted cotton oligonucleotide microarrays for gene expression analysis.

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Spotted cotton oligonucleotide microarrays for gene expression analysis.

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