Dataset features


Application: Gene expression microarray analysis
Number of samples: 20
Release date: Aug 1 2006
Last update date: Mar 16 2012
Access: Public
Dataset link Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei

Experimental Protocol

Total RNA was extracted from pooled stem and leaf samples for each genotype (FLIP94-508C, ICC3996, ILWC245 and Lasseter) 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 according to [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Published in Plant Biotechnology Journal]. 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, 360 images were analyzed from 60 slides, resulting in 18 data points for each time-point of each genotype. 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 1.8-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.








Tristan Coram