For real time PCR analysis, total RNA was extracted from 8 to 20 mg of muscle tissue using a combination Trizol reagent (Life Technologies Inc., Burlington, ON, Canada) and RNeasy mini kit (Qiagen, Toronto, ON, Canada). cDNA was generated from 0.5 μg of total RNA with Superscript III reverse transcriptase (Invitrogen) using random primers. The levels of Fndc5, Pgc1α and Tbp (TATA-binding protein) were determined using TaqMan assays on demand (Invitrogen). Relative RNA abundance was calculated using the ΔΔCT method as we have described previously (O'Neill et al. 2011 (link)) using Tbp as an endogenous control.
Protein abundance was determined using Western blot analysis as we have described previously (O'Neill et al. 2011 (link)) using the following commercially available antibodies: Acetyl-CoA Carboxylase (ACC) (#3676), ACC pSer79 (#3661), AMPK pan α (#2532), AMPK pThr172 (#2532), GAPDH (#5174), all from Cell Signaling Technology, Inc., Danvers, MA and FNDC5 using Irisin (42–112) purified IgG antibody (Phoenix Pharmaceuticals, Burlingame, CA).
Protein abundance was determined using Western blot analysis as we have described previously (O'Neill et al. 2011 (link)) using the following commercially available antibodies: Acetyl-CoA Carboxylase (ACC) (#3676), ACC pSer79 (#3661), AMPK pan α (#2532), AMPK pThr172 (#2532), GAPDH (#5174), all from Cell Signaling Technology, Inc., Danvers, MA and FNDC5 using Irisin (42–112) purified IgG antibody (Phoenix Pharmaceuticals, Burlingame, CA).