Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution.
Journal
Genome research
ISSN: 1549-5469
Titre abrégé: Genome Res
Pays: United States
ID NLM: 9518021
Informations de publication
Date de publication:
11 2019
11 2019
Historique:
received:
15
03
2019
accepted:
20
09
2019
pubmed:
25
9
2019
medline:
6
5
2020
entrez:
25
9
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31548359
pii: gr.250480.119
doi: 10.1101/gr.250480.119
pmc: PMC6836731
doi:
Substances chimiques
DNA, Mitochondrial
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1878-1888Subventions
Organisme : NCRR NIH HHS
ID : C06 RR030414
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA142543
Pays : United States
Organisme : NIA NIH HHS
ID : R01 AG001228
Pays : United States
Informations de copyright
© 2019 O'Hara et al.; Published by Cold Spring Harbor Laboratory Press.
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