Quantitative RT-PCR Analysis of mRNA Expression

Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Isolated RNA was subsequently reverse-transcribed to cDNA using a MMLV Reverse Transcriptase Kit (Bioneer, Daejeon, Korea) and incubation at 37 °C for 60 min followed by incubation at 95 °C for 5 min using GeneAMP^® PCR system 2700 (Applied Biosystems, Foster City, CA, USA). mRNA expression was determined by qRT-PCR as described previously [38 (link)]. Primer sequences used are provided in Table S1. mRNA level of each target gene was normalized to the housekeeping gene, β-actin and expressed as the fold change relative to C2C12 control cells.