Evaluation of mtDNA in Fibroblasts

For the evaluation of mtDNA copy number and deletions in fibroblasts, genomic DNA was isolated from 1 x 10⁶ cells from fibroblasts using the Gentra Puregene Cell and Tissue Kit (Qiagen, Valencia, CA) and following the manufacturer’s recommendations for cell samples. Concentration and purity of DNA was measured at 260 nm and 280 nm on a Tecan infinite M200 Nanoquant (Tecan, Austria). DNA was diluted to 0.63 ng/μl and served as stock DNA template for qPCR. Following DNA extraction, changes in mtDNA copy number and deletions were evaluated by using dual-labeled probes as described in details elsewhere [15 (link), 18 (link)]. Relative mtDNA copy number per cell was assessed by a comparative Ct method, using the following equation: mtDNA/nDNA=2^−ΔCt, where ΔCt=Ct_{mitochondrial}-Ct_nuclear. Each sample was analyzed in triplicates. The ND1 gene copy number was normalized by the single copy nuclear gene (pyruvate kinase) to reflect the mtDNA copy number per cell whereas the ratios of CYTB or ND4 copy number normalized to that of ND1 reflected mtDNA deletions in the major arc.