Nucleic Acids Research, 2003, Vol. 31, No. 11 e61
© 2003 Oxford University Press
Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age
1 Cardiac Surgical Research Unit, Alfred Hospital and Baker Heart Institute, Commercial Road, Prahran, Victoria 3181, Australia, 2 Department of Biochemistry and Molecular Biology, Monash University, PO Box 13D, Clayton, Victoria 3800, Australia and 3 Centre for Molecular Biology and Medicine, Epworth Medical Centre, 185–187 Hoddle Street, Richmond, Victoria 3121, Australia
*To whom correspondence should be addressed. Tel: +61 3 9905 3735; Fax: +61 3 9905 4699; Email: phillip.nagley{at}med.monash.edu.au
Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy ß-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 ± 920, significantly higher than that in skeletal muscle, 3650 ± 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.
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