Preparation of cRNA and hybridization using Affymetrix Gene Chip

Biotin labelled cRNA was prepared using Gene Chip^®3′ IVT Express Labeling Kit (Affymetrix, Santa Clara, CA, USA). All the procedures followed were as per the manufacturer’s protocol (Affymetrix). Briefly, 100 ng of total RNA was used to synthesize double stranded cDNA using an oligo(dT) primer containing the T7 RNA polymerase promoter site provided with GeneChip^®3′ IVT Express Labeling Kit (Affymetrix, Santa Clara, CA). Purification of cRNA was done to remove unincorporated nucleotide triphosphates, salts, enzymes, and inorganic phosphates prior to fragmentation and hybridization onto Human Genome U133 Plus 2.0 Gene Chip expression arrays. Genome arrays were hybridized for 16 hours overnight at 45°C as per the affymetrix protocol. Following washing and staining, the Gene Chips were scanned using the Affymetrix Gene Chip Scanner 3000. The raw data file formats were generated using Gene Chip operating software (GCOS).