Two micrograms of total RNA (from same RNA used for microarrays) was reverse transcribed using the TaqMan Reverse Transcription Reagent kit (Applied Biosystems, Foster City, Calif., USA). An aliquot of the cDNA was amplified for 40 cycles on a GeneAmp 7900HT Sequence Detection System with gene-specific primers designed using the Primer Express software (Applied Biosystems). Sybr Green was used for signal detection. All analyses were carried out in triplicate, and no-template controls and dissociation curves were used to ensure specific amplification. For each primer pair, serial dilutions of a control cDNA were used to determine standard curves, and curves with R2> 0.98 were then used to determine the mRNA levels in individual samples. The expression levels were calculated as a ratio of the mRNA level for a given gene relative to the mRNA level for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same cDNA.
