Quantitative Real-Time PCR and Mitochondrial DNA

Total RNA preparations were extracted with TRIzol reagent (TaKaRa, Japan) from the cells and quantified using a NanoDrop (Thermo Fisher Scientific, United States). One microgram of total RNA was reverse transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative real-time PCR was performed using amplified cDNA, gene-specific primers, and a SYBR Green dye kit (TaKaRa, Japan) using QuantStudio 3 (Thermo Fisher Scientific, United States). GAPDH was used as the housekeeping gene. The relative gene expression was calculated by the 2^{–Δ Δ Ct} method. Mitochondrial DNA content was quantified as described previously (Ulmer et al., 2018). Genomic and mitochondrial DNA was extracted with a genomic DNA extraction kit (BioFlux, China). The relative content of mtDNA was detected by quantitative real-time PCR using mitochondria-encoded NADH dehydrogenase (mt-ND1 or mt-ND2) primers and normalized to β-globin. All primers were purchased from TSINGKE Biological, and the sequences of the primers are listed in Supplementary Table S1.