Quantitative PCR Analysis of mRNA

The analysis of particular mRNA was performed by RT-PCR as described previously (37 (link)). cDNAs were synthesized from 1.5 μg of RNA using the ThermoScript RT-PCR instrument for first-strand synthesis (Invitrogen). Quantitative PCR (qPCR) reactions were performed using a cDNA mix with primers in a final volume of 25 μl in an Applied Biosystems Quant Studio 6-Flex Real-Time PCR instrument (Norwalk, CT, USA). The parameter of the cycle was as follows: 50°C for two minutes, one step of denaturation at 95°C for ten minutes, and 40 cycles of denaturation at 95°C for ten seconds, followed by annealing and elongation at 60°C. The ΔΔCt analysis normalized each transcript’s relative expression level to that of GAPDH. Table S1 of the Supplemental Information presents the primer sequences used for qPCR.