Quantification of Mitochondrial DNA Content

To measure the mitochondrial DNA content, we determined the ratio of one mitochondrial gene to a nuclear gene using quantitative real-time RT-PCR as described previously with some modifications (Pieters et al., 2013 (link)). Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's instructions (Quick-Start Protocol). The concentration of the extracted DNA was measured spectrophotometrically at 260 nm with the NanoDrop 2000 (Thermo Scientific, Wohlen, Switzerland). Afterwards, DNA was diluted in RNase free water to a final concentration of 10 ng × μL⁻¹. The analysis of the mitochondrial cytochrome b and the nuclear pyruvate kinase genes purchased from Microsynth (Balgach, Switzerland) was performed using SYBR Green real-time PCR (Roche Diagnostics, Rotkreuz, Switzerland) on an ABI PRISM 7700 sequence detector (PE Biosystems, Rotkreuz, Switzerland). Relative amounts of nuclear and mitochondrial DNA were determined by comparison of the amplified and quantified DNA from pyruvate kinase and cytochrome b (Primer sequences in Table 1). The amount of mitochondrial DNA was also normalized to citrate synthase activity.