Mitochondrial DNA (mtDNA) quantification

Relative amounts of nuclear DNA and mtDNA were determined by quantitative Real-Time PCR. Differentiated C2C12 myotubes were incubated with Proteinase K overnight in a lysis buffer for DNA extraction by using the DNeasy kit (QIAGEN). Quantitative PCR was performed by using the following primers (mtDNA, forward 5`-CCGCAAGGGAAAGATGAAAGA-3`, reverse 5`-TCGTTTGGTTTCGGGGTTTC-3`; and nuclear DNA, forward 5`-GCCAGCCTCTCCTGATGT-3`, reverse 5`- GGGAACACAAAAGACCTCTTCTGG-3` and FastStart Universal SYBR Green Master mix in a LightCycler 96 (Roche) with a program of 20 minutes at 95°C, followed by 50 to 60 cycles of 15 seconds at 95°C, 20 seconds at 58°C and 20 seconds at 72°C. Mitochondrial DNA content was normalized with nuclear DNA content.