Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution: a focus on aging and cancer

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. Importantly, 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 (ddMDM; droplet digital mitochondrial DNA measurement) 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 hours, 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. Importantly, 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.

Anin situatlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells

“…9 As such, there has been interest in measuring mtDNA copy number (independent of mtDNA mutations) in aging and diseased tissues. [10][11][12][13][14][15][16] In human studies, while there is general agreement that mtDNA copy number declines with age in peripheral blood mononuclear cells, [17][18][19][20] no consensus has been reached regarding mtDNA copy number changes with aging in non-blood related tissue types. 15 This is the case even in skeletal muscle, in which both overall cellular/organ function and mitochondrial function are well-known to decline with age.…”