Quantification of mtDNA Using qPCR (Real-Time PCR)

Mitochondrial mass determination and mtDNA copy number are indicators of mitochondrial content (15). We used qPCR to quantify mtDNA molecules. Briefly, the concentration of purified total DNA was measured using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). The mtDNA (12S) copy number was determined by qPCR and compared to genomic DNA (RNase P), as previously described (22). Briefly, mtDNA was quantified using 125 nM specific primers for the gene 12S (12S-F: 5-CCA CGG GAA ACA GCA GTG ATT-3 and 12S-R: 5-TAT TGA CTT GGG TTA ATC GTG TG-3) and 140 nM of the TaqMan MGB Probe (6'FAM GTG CCA GCC ACC GCG MGENFQ). gDNA copy number was quantified using 1X of the PDARS RNase P Kit (VIC, Thermo Scientific, P/N 4316944) and 1X Universal master mix (Thermo Scientific, USA). mtDNA and gDNA were amplified in a single tube using four 1:5 serial dilutions, beginning with 5 ng/μL of total DNA. All samples, including serial dilutions, were analyzed in triplicate. The amount of mtDNA relative to RNase P was calculated using the following formula: mtDNA/RNase P = 2^{−(CtmtDNA−CtRNAseP)}, where Ct is the threshold cycle.