Quantification of mtDNA Deletions by ddPCR

MtDNA deletions were measured by droplet digital PCR as described previously [63 (link)]. Briefly, total genomic DNA was isolated by phenol/chloroform extraction from previously flash frozen muscle. Ten micrograms were restriction digested with TaqI enzyme and extracted again by phenol/chloroform extraction. Droplet PCR reaction mixture and cycling conditions are described elsewhere [63 (link)]. Reaction droplets were made by applying 20 μL of each reaction mixture to a droplet generator DG8 cartridge (Bio-Rad) for use in the QX100 Droplet Generator (Bio-Rad). The thermally cycled droplets were analyzed by flow cytometry in a QX100TM Droplet DigitalTM Reader (Bio-Rad) for fluorescence analysis and quantification of mutation frequencies. The number of target molecules per droplet was calculated automatically by the QuantaSoft software (Bio-Rad) using Poisson statistics as described elsewhere [63 (link)].