Quantification of RNA Yield by Spectrophotometric Analysis🔗

Absorbance of the sample was checked in 260 and 280 nm. The ratio of A260/A280 was 1.9 which is in an acceptable range.

cDNA and cRNA Synthesis, Hybridization, Scanning, Data Extraction were done.

• a) Single cycle labeling process with cDNA using a T4 promoter—dT24 oligonucleotide as primer and second strand cDNA synthesis and incubation with T4 DNA polymerase has been done and the products were purified using affymetrix clean up module. Biotinylated cRNA is made using affy IVT kit.
• b) cRNA is purified using Qiagen, quantified and then fragmented by incubation at high temperature with Magnesium. Biotinylated cRNA is added to a total hybridization cocktail of 300 microliters and 200 microliters is hybridized to gene chip after adding control oligo-nucleotides. overnight c) Hybridization has been done at 45o C, 17 hours overnight with constant rotation. The hybridization mixture is then removed and the Gene Chips are washed, stained with phycoerythrin-labeled

straptavidin, washed, incubated with biotinylated anti-streptavidin and then restrained with phycoerythrin-labeled straptavidin to amplify the signals.

• d) Arrays are then scanned using dedicated scanner, controlled by GCOS software. Images are examined for defects. The Affy Micro Array Suite 5 (MAS%) algorithm analyzed the hybridization intensity data of gene chip expression probe arrays checked normalized and CEL files were stored for further analysis.
• e) Microarray Data Analysis was done using the latest version software Gene Spring 11.5. The CEL files are imported in Affymetrix Expression workflow using Technology for HG-U133_Plus_2. Summarization: We opted for MAS 5 summarization. It also provides flag information for the probe sets. Baseline to median of all samples was applied for this data. Analysis: A t test unpaired was used to identify the differentially expressing

entities. Fold change analysis at the cut-off of 2 was performed. Hierarchical clustering on the output was performed. Gene Ontology Analysis was done by using fold change output. NCI to find significant pathways was used for pathway analysis.