Quantitative RT-PCR of Muscle Transcripts

Total RNA was isolated from ~20 mg of human skeletal muscle tissue using the acid phenol method [26 (link)]. Total RNA was isolated from C2C12 myotubes as previously described [27 (link)]. RNA quantity was confirmed with the NanoDrop (NanoDrop Technologies, Wilmington, DE, USA). ANT1, Rn18s and GAPDH primers were obtained from Applied Biosystems (Roche, Branchburg, NJ, USA). Real-time quantitative RT-PCR (qRT-PCR) reactions were performed as one-step reactions on the ABI PRISM 7900 real-time PCR system from Applied Biosystems (Nieuwerkerk aan den Ijssel, the Netherlands) as previously described [28 (link)]. SYBR Green was used as the reporter dye. For all assays performed in human samples, GAPDH was the reference gene, and for all assays performed on C2C12 myotubes, Rn18s was the reference gene. All expression data were normalised by dividing the target gene by the reference gene. All qRT-PCR measurements were performed in triplicate.