Dataset features


Application: Gene expression microarray analysis
Number of samples: 16
Release date: Apr 3 2007
Last update date: Mar 19 2012
Access: Public
Chemicals: Fructose, Glycine, Plastoquinone
Dataset link Gene expression profiling of chickpea responses to high-salinity stress

Experimental Protocol

Total RNA was extracted from separately pooled shoot and root tissues for each genotype {CPI 060546 (Tolerant1), CPI 60527 (Susceptible1), ICC 06474 (Tolerant2) and ICC 08161 (Susceptible2)} at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity and quality of the total RNA samples were assessed by OD260/OD280 ratios and gel electrophoresis respectively. Fluorescent-labeled targets were prepared and hybridized to array slides as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. All hybridizations were performed with six technical replicates and three biological replicates, incorporating dye-swapping (i.e. reciprocal labelling of Cy3 and Cy5) to eliminate any dye bias. Overall, 576 images were analyzed from 96 genotype x tissue type x treatment/control x biological replication condition. Slides were scanned at 532 nm (Cy3 green laser) and 660 nm (Cy5 red laser) at 10 µm resolution using an Affymetrix® 428™ array scanner (Santa Clara, CA), and captured with the Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Image analysis was performed using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Quantified spot data was then compiled and transformed using GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA). Data transformations consisted of a local background correction (mean intensity of background was subtracted from mean signal intensity for each spot), omitting flagged spots, LOWESS normalisation of the entire population, creating a Cy5/Cy3 mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. To identify differentially expressed (DE) genes, expression ratio results were filtered to eliminate genes whose 95% confidence interval for mean fold change (FC) did not extend to 2-fold up or down, followed by Students t test with False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression changes versus untreated control were significant at P < 0.05.








Nitin Mantri

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