Samples for gene expression analysis were labeled using Agilent Quick-Amp labeling Kit one-color. Aliquots of 500 ng of each sample were incubated with a reverse transcription mixture at 40 °C and converted to double stranded cDNA primed by oligo (dT) with a T7 polymerase promoter. Synthesized double stranded cDNA was used as template for cRNA generation. The cRNA was generated by in vitro transcription, and the dye Cy3 CTP (Agilent) was incorporated during this step, both carried out at 40 °C. Labeled cRNA was cleaned up, and its quality was assessed using NanoDrop® ND-1000 UV-Vis spectrophotometer. The specific activity determination was based on the cRNA concentration and dye incorporation.
The labeled cRNA samples were hybridized onto an AZ-specific microarray chip, AMADID: 04331034 (link) designed by Genotypic Technology Pvt. Ltd (Bangalore, India). Aliquots of 1650 ng of Cy3-labeled samples were fragmented and hybridized by using the Gene Expression Hybridization kit of Agilent. Hybridization was carried out in an Agilent SureHyb chamber at 65 °C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers, and scanned using the Agilent Scanner, Part Number G2600D. Data extraction from images was performed by using Feature Extraction software of Agilent V-11.5.
The labeled cRNA samples were hybridized onto an AZ-specific microarray chip, AMADID: 04331034 (link) designed by Genotypic Technology Pvt. Ltd (Bangalore, India). Aliquots of 1650 ng of Cy3-labeled samples were fragmented and hybridized by using the Gene Expression Hybridization kit of Agilent. Hybridization was carried out in an Agilent SureHyb chamber at 65 °C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers, and scanned using the Agilent Scanner, Part Number G2600D. Data extraction from images was performed by using Feature Extraction software of Agilent V-11.5.