2.6. Quantification of Mitochondrial DNA

Total DNA (nuclear and mtDNA) was extracted from 10 mg of the mouse liver (the edge of the right lateral lobe) using a DNA-Extran 2 kit (Cat. No. EX-511, Sintol, Russia) in accordance with the protocol of the manufacturer. One nanogram of the total DNA was taken for the reaction. The mtDNA content in the liver tissue was evaluated by PCR, as described [30] and expressed as mtDNA/nuclear DNA ratio. For the assay, we selected the ND4 gene of the mitochondrial genome and the GAPDH gene of the nuclear one. A comparison of ND4 DNA expression relative to GAPDH DNA expression will give a measure of mtDNA copy number to nDNA copy number ratio. Primers for mtDNA and nDNA are presented in Table 1. The real-time PCR was performed on a DTLite5 amplifier (DNA-Technology LLC, Moscow, Russia) using the qPCRmix-HS SYBR reaction mixture (Eurogen, Moscow, Russia), which contained the commonly used fluorescent DNA binding dye SYBR Green II.