RT Journal
A1 O'Hara, Ryan
A1 Tedone, Enzo
A1 Ludlow, Andrew
A1 Huang, Ejun
A1 Arosio, Beatrice
A1 Mari, Daniela
A1 Shay, Jerry W.
T1 Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution
JF Genome Research 
JO Genome Research 
YR 2019 
FD November 01 
VO 29 
IS 11 
SP 1878 
OP 1888 
DO 10.1101/gr.250480.119 
UL http://genome.cshlp.org/content/29/11/1878.abstract 
AB 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.