Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution
Open accessPublished version of a preprint
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.
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A mild gradual age-related reduction of mtDNA copies was observed in stimulated peripheral blood mononuclear cells in a sample ranging in age between 23 and 113 years.
“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].”
From Role of Age-Related Mitochondrial Dysfunction in Sarcopenia (2020) · cites this paper as [37]
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.
“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].”
From Role of Age-Related Mitochondrial Dysfunction in Sarcopenia (2020) · cites this paper as [37]
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References 70 · Cited by 121
- Authors
- O'Hara, R., Tedone, E., Ludlow, A., Huang, E., Arosio, B., Mari, D., & Shay, J. W.
- Affiliations
- The University of Texas Southwestern Medical Center (US)
- University of Milan (IT)
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2019-09-23
- Volume
- 29
- Issue
- 11
- Pages
- 1878-1888
- ISSN
- 1088-9051 (ISSN-L)
- 1549-5469
- PMC
- PMC6836731
- OpenAlex
- W2975207555
- Cited by
- 121
- References
- 70
- Citations by year
- 2026: 14 · 2025: 16 · 2024: 17 · 2023: 16 · 2022: 23 · 2021: 20 · 2020: 14 · 2019: 1
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Institutes of Health (AG01228, C06RR30414)
- National Cancer Institute
- Comprehensive Cancer Center
- Southland Financial Corporation Distinguished Chair in Geriatric Research
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Innovative Microfluidic and Catalytic Techniques Innovation
- Keywords
- mitochondrial DNA copy number, droplet digital PCR, single-cell quantification, tumor progression, cell cycle progression, T cell activation, aging, biomarkers
- MeSH terms
- Adult, Aged, DNA, Mitochondrial, Humans, Lymphocyte Activation, T-Lymphocytes, Polymerase Chain Reaction, DNA Copy Number Variations, Limit of Detection, Single-Cell Analysis
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