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

Abstract

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 beta-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.

Figure 1

Dual-insert plasmid pFM11 showing sites into which specific segments of human mtDNA and nuclear DNA have been inserted. Schematic (not to scale) indicates polylinker region of plasmid pUC18 with sequences bounded by LacI and LacZ′. Representative restriction sites are indicated including insertion sites of mtDNA (part of cytochrome b gene) and nuclear DNA (part of β-globin gene) at SmaI and HincII sites in the original pUC18 plasmid, respectively (the now-destroyed sites are indicated in parentheses).

Figure 2

Representative gel of PCR products of human skeletal muscle DNA extracts used to determine relative abundance of different regions of mtDNA. Gel image shows PCR products from serially diluted inputs of reference DNA extract (1 h old skeletal muscle extract, T1, lanes 1–10) and other human skeletal muscle DNA: tissues T2 (5 weeks), T3 (5 years), T4 (50 years), T5 (84 years) and T6 (90 years). Tissues T2–T6 underwent duplicate analyses (lanes 11–20); the product intensities are similar for each pair, demonstrating a high degree of reproducibility.

Figure 3

Human mtDNA copy number in skeletal muscle as a function of age (n = 29). The line represents a linear regression analysis (R² = 0.01, P = 0.56).

Figure 4

Human mtDNA copy number in right atrium of heart as a function of age (n = 35). In cases where multiple atrial strips were analysed (Table 3) the mean was used. The line represents a linear regression analysis (R² = 0.005, P = 0.70).