Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

Abstract

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

Role of Age-Related Mitochondrial Dysfunction in Sarcopenia

“…MtDNA copy number also varies during aging and might be considered a biomarker that mirrors alterations within the aged human body [34][35][36]. A new quantitative, highly sensitive droplet digital PCR method has allowed observing a mild gradual age-related reduction of the mtDNA copies in stimulated peripheral blood mononuclear cells in a sample ranging in age between 23 and 113 years [37]. Interestingly, centenarians with lower levels of frailty showed a significantly higher number of mtDNA molecules and fewer mtDNA deletions compared with those with more severe frailty [37].…”

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases

“…The droplet digital PCR (ddPCR) using the water-in-oil droplet technique exhibited a higher sensitivity to distinguish rare mutations with a 0.001% sensitivity 23 . It has also been utilized to measure the mitochondrial DNA copy number using eluted nucleotides 24 or lysed cells 25 . Although these assays claimed to provide single cell resolution by harnessing the limiting dilution, the results are based upon the hypothesis that all cells possess the same heterogeneity of mtDNA.…”

Heteroplasmy and Copy Number in the Common m.3243A>G Mutation—A Post-Mortem Genotype–Phenotype Analysis

“…Mitochondria are involved in various cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation and motility, as well as free radical generation [1]. Mostly, mitochondria are called the "powerhouse of the cell" as they provide up to 90% of a cell's adenosine triphosphate (ATP) [2].…”

“…Post-mitotic tissues such as muscle have higher levels of mutant mtDNA, whilst levels in blood decrease significantly over time [8]. The mtDNA is present at hundreds to thousands of copies per cell in a tissue-specific manner [1]. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body [1].…”

“…The mtDNA is present at hundreds to thousands of copies per cell in a tissue-specific manner [1]. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body [1].…”