Abstract
Mitochondria contain multiple copies of the circular mitochondrial genome (mtDNA) that encodes ribosomal RNAs and proteins locally translated for oxidative phosphorylation. Loss of mtDNA integrity, both altered copy number and increased mutations, is implicated in cellular dysfunction with aging. Published data on mtDNA copy number and aging is discordant which may be due to methodological limitations for quantifying mtDNA copy number. Existing quantitative PCR (qPCR) mtDNA copy number quantification methods provide only relative abundances and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA for normalization. We have developed and validated a novel absolute mtDNA copy number quantitation method that uses chip-based digital polymerase chain reaction (dPCR) to count the number of copies of mtDNA and used this novel method to assess the literature discrepancy in which there is no clear consensus whether mtDNA numbers change with aging in skeletal muscle. Skeletal muscle in old mice was found to have increased absolute mtDNA numbers compared to young controls. Furthermore, young Sod1 −/− mice were assessed and show an age-mimicking increase in skeletal muscle mtDNA. These findings reproduce a number of previous studies that demonstrate age-related increases in mtDNA. This simple and cost effective dPCR approach should enable precise and accurate mtDNA copy number quantitation in mitochondrial studies, eliminating contradictory studies of mitochondrial DNA content with aging.
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Acknowledgments
The authors wish to thank the Laboratory for Molecular Biology and Cytometry Research at OUHSC for providing Sanger sequencing assistance and Peter Eckhart for assistance with figure generation. This work was supported by the National Institutes of Health [National Eye Institute R01EY02176, R21EY024520 to WMF, T32EY023202 to DRM]; National Institute on Aging Oklahoma Nathan Shock Center (P30AG050911) to HVR, WMF; the Donald W. Reynolds Foundation [DRM & WMF]; The University of Oklahoma Health Sciences Center Graduate Student Association research grant to [DRM]; and in part by an award from Harold Hamm Diabetes Center at the University of Oklahoma [DRM].
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Masser, D.R., Clark, N.W., Van Remmen, H. et al. Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle. AGE 38, 323–333 (2016). https://doi.org/10.1007/s11357-016-9930-1
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DOI: https://doi.org/10.1007/s11357-016-9930-1