Quantification of Mitochondrial DNA

Total DNA was extracted from the mouse gluteal muscle using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. NADH dehydrogenase 4 (Nd4), D-loop, cytochrome c oxidase I (Cox1), and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) were amplified by realtime PCR using a LightCycler 2.0 (Roche, Mannheim, Germany). Each 10-µl reaction contained 2 mM MgCl₂, 0.5 µM of each primer, 1x Light-Cycler DNA Master SYBR Green I (Roche), and 15 ng of DNA template. All primer sequences are listed in Table 1. The reaction conditions were as follows: denaturation (95℃ for 10 min), amplification for 35 cycles (95℃ for 10 sec; 62℃ for 10 sec; 72℃ for 10 sec), a melting curve program (65℃ to 95℃ with a heating rate of 0.1℃/sec), and a cooling step (40℃). The results are expressed as the ratio of mtDNA to gDNA and compared as previously described [26 (link)].