Fig. 6

Mitochondrial DNA copy number, integrity and complex activity. a Relative mtDNA copy number in fibroblasts of individual I, the parents of individual I and 5 healthy controls. mtDNA copy number was assessed by comparing expression levels of a mitochondrial DNA fragment (D-loop) versus a nuclear encoded gene (B2M). Relative mtDNA copy number was calculated by calculating ?CT (= CT nuclear gene, B2M?CT mitochondrial gene, D-loop) and converting ?CT to relative expression levels (relative expression = 2^?CT) [31]. Results are pooled values from 4 experiments and plotted as average ± SEM. b MtDNA integrity by long-range PCR. Lane 1 and 2 contain DNA ladders (m); 1 kb Plus ladder and (M); HindIII-digested ?DNA fragments ladder, respectively. Lanes 3?6 contain anonymized diagnostic samples, lane 7 contains the sample of individual I, lane 8 the father of individual I and lane 9 the mother of individual I. Lane 10?12 also contain anonymized diagnostic samples. This procedure is a diagnostic test, performed at Clinical Genetics at Maastricht UMC + . No large mtDNA deletions could be observed. c Mitochondrial respiratory complex activity. Enzymatic activity of mitochondrial respiratory complexes I?V were measured using a spectrophotometer in lysates of cultured fibroblasts [33]. Relative values are calculated using the value of Citrate Synthase activity as the reference value. Bars are averages of 2 measurements. This assay is a diagnostic assay, performed at the Translational Metabolic Laboratory of Radboud UMC