Dataset Information

Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.

ABSTRACT: The availability of comprehensive sets of genes has prompted the researchers to carry out systematic collection of gene expression data. RT-PCR has the highest specificity and sensitivity for transcript detection among all available methods. Low throughput, especially when quantitative data are desired, has precluded RT-PCR from genome-wide application. Here we report a PCR-based expression profiling method, introduced amplified fragment length polymorphism (iAFLP), that has the same specificity and sensitivity as RT-PCR and a throughput level comparable to that of DNA-microarray hybridization. In this method, restricted ends of total cDNAs from six sources were ligated with adaptors having various length of short insertions to a common sequence (polymorphic adaptors). Amplification of a pool of these differentially adapted cDNAs with a gene-specific primer and an adaptor primer allows us to quantitate the abundance of any transcript in six mRNA sources. Using three different primer colors this technique allows quantitation of expression of 864 genes across six different sources per day with a single autosequencer, which is comparable to the throughput of microarray analysis in terms of number of genes x number of sources.

SUBMITTER: Kawamoto S

PROVIDER: S-EPMC311005 | biostudies-literature | 1999 Dec

REPOSITORIES: biostudies-literature

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Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.

Kawamoto S S Ohnishi T T Kita H H Chisaka O O Okubo K K

Genome research 19991201 12

The availability of comprehensive sets of genes has prompted the researchers to carry out systematic collection of gene expression data. RT-PCR has the highest specificity and sensitivity for transcript detection among all available methods. Low throughput, especially when quantitative data are desired, has precluded RT-PCR from genome-wide application. Here we report a PCR-based expression profiling method, introduced amplified fragment length polymorphism (iAFLP), that has the same specificity ...[more]

PMID: 10613853

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Project description:BACKGROUND: Rice is highly sensitive to drought, and the effect of drought may vary with the different genotypes and development stages. Genome-wide gene expression profiling was used as the initial point to dissect molecular genetic mechanism of this complex trait and provide valuable information for the improvement of drought tolerance in rice. Affymetrix rice genome array containing 48,564 japonica and 1,260 indica sequences was used to analyze the gene expression pattern of rice exposed to drought stress. The transcriptome from leaf, root, and young panicle at three developmental stages was comparatively analyzed combined with bioinformatics exploring drought stress related cis-elements. RESULTS: There were 5,284 genes detected to be differentially expressed under drought stress. Most of these genes were tissue- or stage-specific regulated by drought. The tissue-specific down-regulated genes showed distinct function categories as photosynthesis-related genes prevalent in leaf, and the genes involved in cell membrane biogenesis and cell wall modification over-presented in root and young panicle. In a drought environment, several genes, such as GA2ox, SAP15, and Chitinase III, were regulated in a reciprocal way in two tissues at the same development stage. A total of 261 transcription factor genes were detected to be differentially regulated by drought stress. Most of them were also regulated in a tissue- or stage-specific manner. A cis-element containing special CGCG box was identified to over-present in the upstream of 55 common induced genes, and it may be very important for rice plants responding to drought environment. CONCLUSIONS: Genome-wide gene expression profiling revealed that most of the drought differentially expressed genes (DEGs) were under temporal and spatial regulation, suggesting a crosstalk between various development cues and environmental stimuli. The identification of the differentially regulated DEGs, including TF genes and unique candidate cis-element for drought responsiveness, is a very useful resource for the functional dissection of the molecular mechanism in rice responding to environment stress.

Dataset Information

Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.

Publications

Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.

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