Transcription profiling of Oryza sativa subtypes Cultivar Nagina-22 (N22) and IR64 subtypes under normal and drougth conditions

High quality RNA was extracted from the whole seedlings (Combined root and leaf samples) using TRI Reagent (Ambion, Inc. USA) and pooled from 12 independent stressed and non-stressed plant samples separately, and treated with DNase-I (QIAGEN GmbH, Germany). Subsequently, RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5 ug of total RNA from each sample in triplicates were reverse-transcribed to double stranded cDNA using the GeneChipﾮ One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipﾮ IVT Labelling Kit (Affymetrix, CA, USA). Twenty microgram of cRNA samples was fragmented and out of which which 7.5 ug cRNA were hybridized for 16 hours at 45C to the Affymetrix GeneChipﾮ Rice Genome Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the Genechipﾮ Fluidics Station 450, the arrays were scanned by the Genechipﾮ 3000 Scanner. The chip images were scanned and extracted using default settings and the CEL files were produced with the Affymetrix GeneChip Operating Software (GCOS 1.2). The resulting .CEL files were imported into the GeneSpring GX 10 (Agilent Technologies Inc, Santa Clara CA) and normalized with the PLIER16 algorithm. The resulting expression values were log2-transformed. Average log signal intensity values of three technical replicates for each sample were used for advance analysis.

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Lenka SK, Katiyar A, Chinnusamy V, Bansal KC