| Mitochondrial DNA analysis
Total DNA was extracted from muscle using phenol/chloroform/isoamyl alcohol (25:24:1) followed by ethanol precipitation. The content of mitochondrial DNA (mtDNA) was calculated using real time quantitative PCR by measuring the threshold cycle ratio (∆Ct) of a mitochondrial-encoded gene (COX1, fwd 5’-ACTATACTACTACTAACAGACCG-3’, rev. 5’-GGTTCTTTTTTTCCGGAGTA-3’) versus a nuclear-encoded gene (cyclophilin A, fwd 5’-ACACGCCATAATGGCACTGG-3’, rev. 5’-CAGTCTTGGCAGTGCAGAT-3’).
Real time quantitative RT-PCR analysis
Total RNA was extracted with the Trizol Reagent (Invitrogen). The level of target mRNAs was measured by RT followed by real-time PCR using a LightCycler (Roche). A standard curve was systematically generated with six different amounts of purified target cDNA and each assay was performed in duplicate (34). We measured hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA as a reference gene so that the results are expressed as a ratio referred to the expression of HPRT. Primer sequence and RT-qPCR conditions are available upon requested.
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