Real-time PCR

mRNA expression of genes was studied using TaqMan qPCR primers/probes according to manufacturer’s instructions. In short, a standard curve was prepared by pooling equal volume of cDNA from each sample that was then serially diluted in to 6 standards. Standards, negative controls and unknown samples were run in duplicate in a 96-well PCR plate. The total reaction volume was 10 μl consisting of LuminoCt qPCR ready mix (Sigma-Aldrich, USA), TaqMan Primer/Probe (Applied Biosystems, Life technologies, USA), water and cDNA. The cycling condition used was as follows: at 95 °C for 1 s and at 60 °C for 20 s for 40 cycles in addition to one step initialization at 95 °C for 20 s in ABI 7900HT Fast Real-Time PCR system (Applied Biosystems). Then, relative quantities were recorded for each well and normalized to the expression of housekeeping gene, GAPDH.