Quantification of Mitochondrial DNA Content

DNA was extracted from frozen Liver tissue (20 mg) using DNeasy Blood & Tissue kit (#69504, Qiagen). DNA quality and quantity were estimated using a Nanodrop spectrophotometer (Thermo Scientific). To estimate the amount of mtDNA relative to nDNA, we used two sets of primers encoding a mitochondrial gene (Mt_Nd1 and Mt_Nd6) and a nuclear gene Tubulin (Tuba1) (24 (link)). iTaq universal SYBR® Green supermix (BioRad, USA) and 50 ng of DNA was used and amplification was conducted using CFX Connect Real-time qPCR system (Bio Rad, USA). The relative mtDNA content was calculated using ΔΔCT method (25 (link)). Accuracy of the target amplicon was checked with a melting curve, and each sample was read in triplicate.