Quantification of Mitochondrial DNA Content

qPCR was used to amplify mtDNA and nucDNA as described before7 (link)15 . Briefly, the total DNA of cultured neurons was extracted and purified using the genomic DNA extraction kit (Qiagen Sciences, CA, USA). The total DNA derived from neurons in one well from 12-well plates was added to the polymerase chain reaction (PCR) mixture with GoTaq Flexi DNA Polymerase (Promega, WI, USA). The PCR products were separated by a 1% agarose gel and stained with ethidium bromide (0.5 mg/mL). The images were acquired by scanning with a Fuji LAS 3000 densitometer (Fuji, Tokyo, Japan). The densities of the mtDNA and nucDNA bands were calculated using ImageJ software. The density ratio of mtDNA to nucDNA was used to determine the relative mtDNA content and mitochondrial mass.