Mitochondrial DNA (mtDNA)

The frozen cardiac muscle was homogenized and the DNA dissolved in 100 μl of Tris-EDTA. Five μl of a 50 times DNA dilution was used for PCR amplification with QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) containing 0.5 μM of each primer in a total volume of 25 μl. Levels of mtDNA and genomic DNA (gDNA), as determined by albumin content, was assessed by real-time PCR using a Biorad IQ real time PCR machine (Stratagene, La Jolla, CA, USA), as previously described (20). The copy number of mtDNA and gDNA was used as an estimate of mitochondrial concentration in cardiac muscle (21).