Quantitative real-time PCR
Total RNA was extracted from tissues and C2C12 myotubes using RNeasy kits (Qiagen, USA), reverse transcribed using High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific, USA) before real time PCR analysis using QuantStudio 7™ (Thermo Fisher Scientific, USA). All reactions were performed in triplicate using Taqman Fast Advanced Master Mix and pre-developed gene expression assays (Table 1) except for Igf1-eb(Thermo Fisher Scientific) and data obtained were normalized against GAPDH as reference gene prior to analysis using the delta delta CT method as previously described (Vlahos, Stambas, Bozinovski, Broughton, Drummond & Selemidis, 2011).