2.8. Quantification of Mitochondrial DNA

Total DNA (nuclear and mtDNA) was extracted from 10 mg of heart tissue using DNA-Extran 2 kit (Sintol, Moscow, Russia) in accordance with the protocol of the manufacture; 1 ng of the total DNA was taken for the reaction. Evaluation of mtDNA content in cardiac tissue was performed by PCR as described [26] and expressed as mtDNA/nuclear DNA ratio. For our assay, we selected the ND4 gene of the mouse mitochondrial genome and GAPDH, which is a nuclear-encoded gene. 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 with a DTLite5 amplifier (DNA-Technology LLC, Moscow, Russia) using the qPCRmix-HS SYBR reaction mixture (Evrogen, Moscow, Russia), which contained a commonly used fluorescent DNA binding dye SYBR Green II.