Quantitative Real-Time PCR Analysis of Gene Expression

Total RNA was extracted from the frozen tissues using TRIzol reagent (Invitrogen), and a quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as described previously [6 (link)]. The real-time PCR protocol consisted of one cycle at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s using the primers for Nppb (Mm01255770_g1; Applied Biosystems), Ppara (Mm00440939_m1; Applied Biosystems), and Ppargc1 (Mm01208835_m1; Applied Biosystems). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH (Mm99999915_g1; Applied Biosystems).