Analysis of mtDNA/nDNA Ratio in Mice

Abstract

Mitochondrial DNA (mtDNA) lacks the protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. mtDNA depletion compromises the efficient function of cells and tissues and thus impacts negatively on health. Here, we describe a brief and easy protocol to quantify mtDNA copy number by determining the mtDNA/nDNA ratio. The procedure has been validated using a cohort of young and aged mice. © 2017 by John Wiley & Sons, Inc.

Keywords: 16SrRNA; ND1; mtDNA; qPCR.

Figure 1. Mouse mitochondrial DNA

The mouse mitochondrial DNA (mtDNA) comprises 16,295 bp. MtDNA forms an inner light strand (L-strand) and an outer heavy strand (H-strand) that encodes different genes. The position of gene names reflects the strand where they are encoded (inside: L-strand: outside: H-strand). Colors identify the different genes: rRNAs (purple), tRNAs (yellow), proteins encoding subunits of the OXPHOS complexes I (blue), III (green), IV (orange) and V (red). 16S rRNA and ND1 genes are located in the stable part of mtDNA, where no deletions have been reported.

Figure 2. Box plot showing the relative mtDNA levels in young and old C57BL/6J mice

Relative quantification was performed on seven young (2 months) and five old (24 months) samples in triplicates using qPCR by amplification of ND1 and 16S genes belonging to stable part of mtDNA, and normalised against hexokinase gene (HK). The decrease in mtDNA/nDNA ratio in old samples is indicative of the decrease in mtDNA copy number with age. Data are represented as box-and-whisker plot. Statistical differences were calculated using Student’s t-test, *** P < 0.001.