Mitochondrial DNA (mtDNA) Copy Number Quantification

mtDNA copy number was determined as a marker for mitochondrial abundance using quantitative real-time PCR (qPCR) method as we previously reported.²³ Briefly, total DNA was isolated by using TIANamp Genomic DNA Kit (TIANGEN, China) according to the manufacturer’s instruction. The concentration of DNA in the extracts was measured by a NanoDrop 2000 Spectrophotometer (Thermo Scientific, USA). DNA primers were designed to detect ND1 (5ʹ-GGAGTAATCCAGGTCGGT-3ʹ and 5ʹ-TGGGTACAATGAGGAGTAGG-3ʹ) as a maker for mtDNA and GAPDH (5ʹ-AAGGTGGAGGAGTGGGTGT-3ʹ and 5ʹ-TCAAGAAGGTGGTGAAGCAG-3ʹ) for nuclear DNA. Reactions were performed on LightCycler 480 II real-time PCR machine (Roche, Switzerland) with SYBR Green I Master (Roche). The reaction conditions were 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 30 s at 60°C; 1 cycle of 5 s at 95°C and 60 s at 60°C; 1 cycles of 30 s at 50°C. Relative amount of mtDNA copy number was calculated by 2^−ΔΔCt method.