Quantifying 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 [26 (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. The analysis of the mitochondrial ND1 and the nuclear 36B4 genes was performed using SYBR Green real-time PCR (Roche Diagnostics, Rotkreuz, Switzerland) on an ABI PRISM 7700 sequence detector (PE Biosystems, Rotkreuz, Switzerland). Quantification was performed using the comparative-threshold cycle method. The list of primers purchased from Microsynth (Balgach, Switzerland) and used in this study can be found in S1 Table.