BMC Genomics 2009, 10:415 http://www.biomedcentral.com/1471-2164/10/415 sequence in common in their BLAST reports were com- bined into a putative gene family as described. [109]. Identification of SNPs For the identification of SNPs, the ace file output of CAP3 programme was used as input to the PolyBayes SNP detec- tion program along with the base values assigned by Phred for each of the contigged sequences. Perl scripts were used to parse the PolyBayes output file. The cutoff value for the probability of SNP was put at 0.99. Only base change mutations were considered in order to avoid any discrepancy in the results due to any error in the align- ment. The SNPs were further divided into high-quality if minimum of two sequences from each genotype showed the same base change and low-quality if minimum of two sequences from one genotype and one from the other showed the same base change. Construction of cDNA microarray The cDNA microarray setup consisted of 8098 probes cor- responding to 2013 unigenes. The cDNA clones from both susceptible and resistant libraries were amplified by performing colony PCR (1× PCR buffer, 1.5 mM MgCl2, 5 units Taq DNA polymerase [Applied biosystems, USA], 0.5 mM dNTPs [Fermentas, Canada], 0.2 M M13 primer [Sigma], 20 ng of template DNA) in 96 well format. The PCR amplified products were purified using Perfectprep PCR clean up kit as per manufacturer's instructions (Eppendorf, Germany). The quality of the PCR products was checked on 1.0% agarose gels. Five microlitres of the PCR products were reorganized on the 384-well printing plates with 5 l of 100% DMSO and printed in duplicates on the poly-L-lysine coated slides (Sigma) using a high throughput arrayer (Arrayer ESI, SDDC2) followed by UV cross linking. The spiking and other controls such as DMSO, SSC, water and previously cloned chickpea genes like 18S rRNA and actin were also printed at different locations in the chickpea microarray which were used as negative and positive controls. Sample preparation, labeling and microarray hybridization In order to find out the differential gene expression during chickpea-Fusarium incompatible interaction, root tissue sample were collected from Fusarium infected WR 315 seedlings after 24 h of infection. Water treated WR 315 plants were taken as control. Two biological replicates were done. Total RNA was isolated using Trizol reagent (Invitrogen, CA). 6 g of the total RNA was used for cDNA synthesis using indirect TSA labeling and detection kit Micromax (PerkinElmer, Boston, MA). The RNA isolated from uninfected and infected tissue was labeled with flourscein and biotin, respectively. The labeled cDNA was purified using microcon YM 100 columns (Millipore, Bedford, MA). The purified flourscein and biotin labeled cDNAs were mixed and hybridized to the microarray slides in hybridization chambers (Corning, USA) at 65°C water bath for 16 hours. The slides were washed for 10 minutes in 0.5× SSC and 0.01%SDS, 10 minutes in 0.06× SSC and 0.01%SDS and for 5 minutes in 0.06× SSC. The slides were subsequently processed for replacement of flourscein with Cy3 and biotin with Cy5 as per manufac- turer's instructions. The experiment was carried out in duplicate. Scanning and data analysis Micraoarrays were scanned using Scan array 5000 scanner (PerkinElmer, MA) to produce two separate tiff images. Spot finding and quantification of the spots were done by using Scan array express software (PerkinElmer, MA). Spots appearing bad due to poor morphology, high local background and bubbles were flagged off and were excluded from further analysis. Spots with both channel intensities less than 500 were also filtered out. Spots were quantified using an adaptive method. Avadis software (PerkinElmer, MA) was used for further data transforma- tion which consisted of background correction and nor- malization. For background correction, local background intensity of each spot was subtracted from its foreground intensity value. Due to non linearity of the data, intensity dependent Lowess normalization was applied. Cy5/Cy3 signal ratio was also calculated. Although, indirect TSA labeling was performed to generate the flourscent labeled probe that obviates the need for control dye swap experi- ments, initially dye swap experiment was performed for checking the reciprocity of the gene signals that showed high correlation between the original and dye swap slides. Cross slide one class t-test with Benjamini and Horchberg FDR multiple correction was performed on the four repli- cate data points for each clone. P-value of 0.05 and fold induction of 2.5 was used as a limit for statistically signif- icant differences in the expression. The resulting differen- tially expressed gene list was uploaded for hierarchical clustering. RNA gel blots Validation of the microarray results was done by northern analysis of a few transcripts. For this, total RNA was extracted from root tissue of water treated and Fusarium challenged plants collected at 24 hours after treatment using trizol reagent (Invitrogen). 20 g of RNA was dena- tured in 50% formamide, 17% formaldehyde, and 10% MOPS buffer (200 mM MOPS, pH7.0, 50 mM sodium acetate and 1 mM EDTA) at 65°C for 5 min and was sep- arated on 1.2% agarose gel containing 22 M formalde- hyde in MOPS buffer, transferred to GenScreen plus membrane (Hybond-N, Amersham Biosciences) by downward capillary blotting in 20× SSC. The DNA probes were prepared by random primer labeling and purified by passing through Sephadex G-50 column as described in Page 17 of 21 (page number not for citation purposes)