Quantitative reverse transcription PCR

Total RNA was extracted from muscle tissues using TRIzol reagent. cDNA was synthesized using the EvoM-MLV kits. Quantitative reverse transcription-PCR (RT-qPCR) was performed using 2X SYBR Green qPCR Master Mix (K1070-500, APExBIO, US) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) following the manufacturer’s protocol and analyzed using the 2^–ΔΔCt method. The following optimized thermal conditions were used: 95°C for 30 s, 95°C for 5 s, and 40 cycles at 60°C for 5 s. The levels of mRNA were normalized to endogenous GAPDH, and the expression of target genes was analyzed using the 2^–ΔΔCt method. The experiments were repeated three times independently. The primer sequences used in this study are listed in Table 2.

Primers used for quantitative reverse transcription-PCR.