Measuring mitochondrial DNA

Mitochondrial and nuclear cytosolic fractions were isolated by differential centrifugation, and mtDNA and nuclear DNA were quantified as previously described⁵⁶. Briefly, liver tissues were gently homogenized in 1 ml isolation buffer (225 mM mannitol, 75 mM sucrose, 10 mM 3-(N-morpholino) propanesulfonic acid, 1 mM ethylene glycol tetra acetic acid, and 0.5% bovine serum albumin; pH 7.2). The extracts were then centrifuged at different speeds to obtain cytosolic- and mitochondrial-enriched fractions. For DNA quantification, RNA from the mitochondrial and cytosolic fractions was removed by treatment with RNase A (Thermo Scientific, UK), and the DNA content was determined spectrophotometrically (Nanodrop ND-1000). The DNA concentration in the mitochondrial fraction was normalized to that in the cytosolic fraction to obtain the mtDNA/nuclear DNA ratio.