RNA Extraction and Real-Time PCR Analysis

Total RNA was extracted from frozen muscle samples using PureLink™ RNA Mini Kit (Invitrogen Canada, Burlington, ON) or Trizol (Life Technologies) following the manufacturer’s instructions. Quantification and purity of RNA were assessed using the A260/A280 absorption method. Total RNA (2 μg or 400 ng) was reverse transcribed using a Superscript II® Reverse Transcriptase Kit and random primers (Invitrogen Canada, Burlington, ON) or the SupersCript IV (Life technologies). Real‐time PCR detection of mRNA expression was performed using a Prism® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) or a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) with SYBR Green chemistry (Power-UP SYBR®Green PCR Master Mix, Applied Biosystem). Cycle threshold (CT) values were obtained for each target gene. ΔCT values (normalized gene expression) were calculated as CT of the target gene minus CT of the geometric means of three housekeeping genes (Cyclophilin, Gapdh, β-Actin, and/or 18 S), unless otherwise indicated in the figure legends. Relative mRNA level quantifications of target genes were determined using the threshold cycle (ΔΔCT) method, as compared to Ctrl. The primer sequences for all genes are found in Supplementary Table S5.