4.8. Determination of Mitochondrial DNA Copy Number

RT-PCR analysis was performed to determine the mtDNA copy number of the experimental groups as described by Fuke and collaborators [95] with slight modifications. Relative quantification of mtDNA levels was determined by the ratio of the mitochondrial ND1 (mt-Nd1) gene to the single-copy, nuclear-encoded beta-2-microglobulin (β-2MG) gene. Reactions were carried out in an iQ5 system (Bio-Rad), and efficiency of the reactions was determined for the selected primers using serial dilutions of DNA samples. Primer concentration and annealing temperature were optimized, and the specificity of the amplicons was determined by melting curve analysis. The reactions mixture consisted of Maxima SYBR Green qPCR Master Mix (Fermentas -Thermo Fisher Scientific, Rockford, IL, USA), sense and antisense primers (Table 2), and 20 ng of DNA. Each reaction was run in triplicate to calculate relative mtDNA copy number. Ct values of all samples were within the linear range. Ct value differences were used to quantify mtDNA copy number relative to the beta-2-microglobulin gene with the following equation: Relative copy number = 2 ΔCt, where ΔCt is Ctβ2MG -CtND1.

Oligonucleotides and Cycling Conditions for qPCR Amplification of ND1 and β-2MG.

Abbreviations: AT—annealing temperature; C—Number of cycles of amplification.