Quantitation of mtDNA Copy Number

Total DNA extraction was performed with a DNA kit (Tiangen Biotech). The quantitation of the mtDNA per nuclear genome used in this study was performed as previously described33 (link)34 (link). In brief, with real-time PCR analysis, the NADH dehydrogenase subunit 1 (ND1), which represents the mtDNA, was normalized to the Pecam gene on chromosome 6, which represents nuDNA. The synthetic oligonucleotide primer sequences for ND1 are 5′-GTC CTC CTA ATA AGC GGC TCC T-3′ (coding sense) and 5′-GAA TGG TCC TGC GGC GTA TTC-3′ (coding antisense), and the sequences of Pecam are 5′-CTA TGG CGG ACA CCT TCC TG-3′ (coding sense) and 5′-TTC TAG GCC TTG GGT GGT CT-3′ (coding antisense). Real-time PCR was performed using SYBR Green dye (Thermo Scientific) with the Eppendorf Realplex PCR system. The relative copy number differences were quantified by analysing the difference in threshold amplification between mtDNA and nuDNA (ΔΔCt method).