Gastrocnemius Muscle RNA Extraction and qRT-PCR

Isolation of total RNA from the gastrocnemius (GA) was performed using an RNeasy Fibrous Tissue Mini Kit (Qiagen Catalog # 74704) according to the manufacturer’s instructions. Skeletal muscle RNA was treated with an RNase-free DNase kit (RNase-Free DNase Set, Qiagen Catalog # 79254) in column according to the manufacturer’s instructions. First strand cDNA was synthesized using 1 μg of purified RNA and a commercially available kit (iScript^TM cDNA synthesis, Bio-Rad Catalog # 170-8891). Quantification of mRNA expression was performed similar to as previously described (Hindi and Kumar, 2016 (link); Fong et al., 2012 (link)) using the SYBR Green dye method on a 7300 Real-Time PCR system (Applied Biosystems) using gene-specific primers, detailed in Supplementary Table 1.