Research Paper Volume 8, Issue 12 pp 3468—3485

Figure 7. Total mtDNA copy number is significantly increased in aged skeletal muscle fibers. NMR quadriceps muscle fibers with normal enzyme activity (such as Cells #11 and #12) were identified in COX-stained sections (A), correlated with SDH-stained sections (B), and laser-capture micro-dissected (C). mtDNA copy number was quantified by real-time PCR-based amplification of various regions of the mitochondrial genome. Total copy number in these cells, normalized to cell area, increased significantly in aged animals compared with young adult animals in all three regions quantified: ND4 (D, p<0.01), ND1 (E, p<0.05), and DLOOP (F, p<0.01). Homogenate tissues were subjected to real time PCR to compare the copy number of DLOOP with that of a single-copy nuclear gene, transthyretin (TTR). This normalized index, which gives the number of mitochondrial DNA copies per nucleus, also significantly increases with age (G). The few COX-deficient cells observed in aged NMR skeletal muscle (such as Cell #7) were laser-capture micro-dissected from SDH-stained serial sections and subjected to multiplexed real-time PCR to quantify the ratio of ND4 (a commonly deleted region) with DLOOP (the control region). COX-deficient cells from aged quadriceps muscle have a significantly lower ND4/DLOOP index than normal cells in aged quadriceps muscle (H).