The Genoplante microarray for wheat [S22-40]
2003
Sarda, Xavier | Sighar, Karim | Giardella, Dea | James, Evelyne | Bourgeois, Emmanuelle | Andrieu, Frédéric | Gautier, Marie-Françoise | Joudrier, Philippe | De Rose, Richard
We choose to study the wheat transcriptome using cDNA parrays. During the phase I of Genoplante, we generated 15 libraries from different tissues and sequenced 100 000 ESTs. Using the contigs generated by Genoplante info, we selected one unique clone per contig to constitute a unigene set. This set contained 27 520 sequences. Viable clones for each unigene were re-arrayed in 384 well plates and amplified by PCR. PCR products were purified, quantified and the DNA concentration was equalized. Finally, the quality of all PCR reactions was analyzed by agarose gel. For array analysis, PCR products were spotted on glass slides in duplicate. Hybridization were performed starting from 180 pg of total RNA by doing a dye swap experiment in triplicate. In addition, RNA amplification can be used and allow one to synthesize fluorescent probes from as little as 0.5 pg total RNA. An informatic routine was developed to control the quality of the raw data and chose a method of normalisation for use during the data acquisition step. Statistical analysis based on the 12 datum point per gene per condition were used as the basis for identification of candidate genes. Biological materials have been prepared with different abiotic situations (thermal and hydric stresses during grain development). They will be used as the source materials for various comparisons done using these mcroarrays for the identification of which genes are specifically induced under these stress conditions. (Texte intégral)
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