Quantifying Mitochondrial DNA in Flies

The total genomic DNA from 30 adult male flies was extracted using a DNeasy Tissue Kit according to the manufacturer’s instructions (3 replicates). The mitochondrial DNA was quantified relative to nuclear DNA by the ratio of amplicons of cytochrome oxidase subunit I (COI) to amplicons of glyceraldehyde 3- phosphate dehydrogenase (GAPDH) in quantitative real-time PCRs. Quantitative PCR (qPCR) was performed using the QuantStudio™ 1 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). At least three replicates were established in each group. The data are presented as mean ± SEM. The qPCR assays were performed with a 60 °C annealing temperature and at 40 cycles. The sequences for the COI and GAPDH were as follows: COI forward 5′-GAA TTA GGA CAT CCT GGA GC-3′, COI reverse 5′-GCA CTA ATC AAT TTC CAA ATC C-3′, GAPDH forward 5′-GAC GAA ATC AAG GCT AAG GTC G-3′, GAPDH reverse 5′-AAT GGG TGT CGC TGA AGA AGT C-3′ [54 (link)].