The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number

doi:10.1016/j.ymeth.2010.01.033

Methods

Volume 51, Issue 4, August 2010, Pages 444-451

https://doi.org/10.1016/j.ymeth.2010.01.033 Get rights and content

Abstract

The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA repair, and describe methodological changes implemented in recent years. We present QPCR assay primers and reaction conditions for five species not previously described in a methods article: Caenorhabditis elegans, Fundulus heteroclitus, Danio rerio, Drosophila melanogaster, and adenovirus. Finally, we illustrate the use of the assay by measuring repair of ultraviolet C radiation-induced DNA damage in the nuclear but not mitochondrial genomes of a zebrafish cell culture.

Section snippets

Theory/rationale

QPCR¹, also called long amplicon PCR (LA-PCR) and long extension PCR (LX-PCR), measures gene-specific damage by quantifying the decrease in amplification of DNA extracted from sample cells of organisms following genotoxin exposure. This assay is based on the principle that induced DNA damage impedes the progression of the DNA polymerase when analyzed by

Protocol

Protocols for this assay have been published previously [2], [14], [26]. Here, we focus on updates and changes made to the assay in recent years.

Example of derived data

As an example, we present data from an experiment in which the QPCR method was adapted to the zebrafish model. Here, we are able to determine the presence of DNA damage and repair in a zebrafish cell line. Zebrafish embryonic fibroblasts (ZF-4 cell line, ATCC # CRL-2050) were cultured at 28 °C (5% CO₂) in Dulbecco’s MEM with 15% FBS and 0.1 mg/mL gentamicin as described [32] and exposed to various doses of ultraviolet C radiation (UVC). DNA was then isolated using the Qiagen Tissue Extraction

Summary

The QPCR assay is a well-validated and sensitive protocol that specifically measures DNA damage in various mitochondrial and nuclear DNA regions in a variety of species, thus permitting investigation of mtDNA and nucDNA damage and repair. This is a particularly useful method in investigating mtDNA damage since differential separation of the mitochondrial DNA is not required, and because very small amounts of DNA can be analyzed. Thus, this method lends itself to biomarker-type work in humans,

Acknowledgments

This research was supported by NIH P42 ES10356 (to RTD) and NIH R21 NS065468 (to JNM); the funding sources played no role in the research related to this publication, nor in the writing. We thank Elwood Linney for providing the ZF-4 cell lines and for cell culture advice, Bennett Van Houten for support during the adaptation of the assay to D. melanogaster, and Amanda Smith for editing suggestions.

References (37)

S. Ayala-Torres et al.
Methods
(2000)
W.A. Boyd et al.
Mutat. Res.
(2010)
J.H. Santos et al.
J. Biol. Chem.
(2003)
D.E. Sawyer et al.
Mutat. Res.
(2003)
J. Termini
Mutat. Res.
(2000)
D. Jung et al.
Aquat. Toxicol.
(2009)
D. Jung et al.
Comp. Biochem. Physiol. C Toxicol. Pharmacol.
(2009)
D. Chandrasekhar et al.
Mutat. Res.
(2000)
C.S. Lassiter et al.
Gene
(2002)
K.L. Willett et al.
Comp. Biochem. Physiol. C Toxicol. Pharmacol.
(2001)

W.M. David et al.

Comp. Biochem. Physiol. C Toxicol. Pharmacol.

(2004)

E.G. Notch et al.

Aquat. Toxicol.

(2009)

M. Ponti et al.

Nucleic Acids Res.

(1991)

J.N. Meyer et al.

Genome Biol.

(2007)

E.C. Friedberg et al.

DNA Repair and Mutagenesis

(2006)

P.C. Hanawalt et al.

Nat. Rev. Mol. Cell Biol.

(2008)

A.C. Haugen et al.

PLoS Genet.

(2010)

F.M. Yakes et al.

Proc. Natl. Acad. Sci. USA

(1997)

Cited by (125)

A state-of-the-science review of using mitochondrial DNA copy number as a biomarker for environmental exposure
2024, Environmental Pollution
Mitochondria are bioenergetic, biosynthetic, and signaling organelles in eukaryotes, and contain their own genomes, mitochondrial DNA (mtDNA), to supply energy to cells by generating ATP via oxidative phosphorylation. Therefore, the threat to mitochondria’ integrity and health resulting from environmental exposure could induce adverse health effects in organisms. In this review, we summarized the association between mtDNA copy number (mtDNAcn), and environmental exposures as reported in the literature. We conducted a literature search in the Web of Science using [Mitochondrial DNA copy number] and [Exposure] as two keywords and employed three selection criteria for the final inclusion of 97 papers for review. The consensus of data was that mtDNAcn could be used as a plausible biomarker for cumulative exposures to environmental chemical and physical agents. In order to furtherly expand the application of mtDNAcn in ecological and environmental health research, we suggested a series of algorithms aiming to standardize the calculation of mtDNAcn based on the PCR results in this review. We also discussed the pitfalls of using whole blood/plasma samples for mtDNAcn measurements and regard buccal cells a plausible and practical alternative. Finally, we recognized the importance of better understanding the mechanistic analysis and regulatory mechanism of mtDNAcn, in particular the signals release and regulation pathways. We believe that the development of using mtDNAcn as an exposure biomarker will revolutionize the evaluation of chronic sub-lethal toxicity of chemicals to organisms in ecological and environmental health research that has not yet been implemented.
Mitochondrial dysfunction and oxidative stress contribute to cross-generational toxicity of benzo(a)pyrene in Danio rerio
2023, Aquatic Toxicology
The potential for polycyclic aromatic hydrocarbons (PAHs) to have adverse effects that persist across generations is an emerging concern for human and wildlife health. This study evaluated the role of mitochondria, which are maternally inherited, in the cross-generational toxicity of benzo(a)pyrene (BaP), a model PAH and known mitochondrial toxicant. Mature female zebrafish (F0) were fed diets containing 0, 12.5, 125, or 1250 μg BaP/g at a feed rate of 1% body weight twice/day for 21 days. These females were bred with unexposed males, and the embryos (F1) were collected for subsequent analyses. Maternally-exposed embryos exhibited altered mitochondrial function and metabolic partitioning (i.e. the portion of respiration attributable to different cellular processes), as evidenced by in vivo oxygen consumption rates (OCRs). F1 embryos had lower basal and mitochondrial respiration and ATP turnover-mediated OCR, and increased proton leak and reserve capacity. Reductions in mitochondrial DNA (mtDNA) copy number, increases in mtDNA damage, and alterations in biomarkers of oxidative stress were also found in maternally-exposed embryos. Notably, the mitochondrial effects in offspring occurred largely in the absence of effects in maternal ovaries, suggesting that PAH-induced mitochondrial dysfunction may manifest in subsequent generations. Maternally-exposed larvae also displayed swimming hypoactivity. The lowest observed effect level (LOEL) for maternal BaP exposure causing mitochondrial effects in offspring was 12.5 µg BaP/g diet (nominally equivalent to 250 ng BaP/g fish). It was concluded that maternal BaP exposure can cause significant mitochondrial impairments in offspring.
Emerging methods for and novel insights gained by absolute quantification of mitochondrial DNA copy number and its clinical applications
2022, Pharmacology and Therapeutics
The past thirty years have seen a surge in interest in pathophysiological roles of mitochondria, and the accurate quantification of mitochondrial DNA copy number (mCN) in cells and tissue samples is a fundamental aspect of assessing changes in mitochondrial health and biogenesis. Quantification of mCN between studies is surprisingly variable due to a combination of physiological variability and diverse protocols being used to measure this endpoint. The advent of novel methods to quantify nucleic acids like digital polymerase chain reaction (dPCR) and high throughput sequencing offer the ability to measure absolute values of mCN. We conducted an in-depth survey of articles published between 1969 -- 2020 to create an overview of mCN values, to assess consensus values of tissue-specific mCN, and to evaluate consistency between methods of assessing mCN. We identify best practices for methods used to assess mCN, and we address the impact of using specific loci on the mitochondrial genome to determine mCN. Current data suggest that clinical measurement of mCN can provide diagnostic and prognostic value in a range of diseases and health conditions, with emphasis on cancer and cardiovascular disease, and the advent of means to measure absolute mCN should improve future clinical applications of mCN measurements.
Association between oxidized nucleobases and mitochondrial DNA damage with long-term mortality in patients with sepsis
2022, Free Radical Biology and Medicine
Sepsis not only leads to short-term mortality during hospitalization, but is also associated with increased mortality during long-term follow-up after hospital discharge. Metabolic stress during sepsis may cause oxidative damage to both nuclear and mitochondrial DNA (mtDNA) and RNA, which could affect long-term health and life span. Therefore, the aim of this study was to assess the association of sepsis with oxidized nucleobases and (mt)DNA damage and long-term all-cause mortality in septic patients.
91 patients with sepsis who visited the emergency department (ED) of the University Medical Center Groningen between August 2012 and June 2013 were included. Urine and plasma were collected during the ED visit. Septic patients were matched with 91 healthy controls. Death rate was obtained until June 2020.The degree of oxidation of DNA, RNA and free nucleobases were assessed in urine by mass-spectrometry. Lipid peroxidation was assessed in plasma using a TBAR assay. Additionally, plasma levels of mtDNA and damage to mtDNA were determined by qPCR.
Sepsis patients denoted higher levels of oxidated DNA, RNA, free nucleobases and lipid peroxidation than controls (all p < 0.01). Further, sepsis patients displayed an increase in plasma mtDNA with an increase in mtDNA damage compared to matched controls (p < 0.01). Kaplan meier survival analyses revealed that high degrees of RNA- and nucleobase oxidation were associated with higher long-term all-cause mortality after sepsis (p < 0.01 and p = 0.01 respectively). Of these two, high RNA oxidation was associated with long-term all-cause mortality, independent of adjustment for age, medical history and sepsis severity (HR 1.29 [(1.17–1.41, 95% CI] p < 0.01).
Sepsis is accompanied with oxidation of nuclear and mitochondrial DNA and RNA, where RNA oxidation is an independent predictor of long-term all-cause mortality. In addition, sepsis causes mtDNA damage and an increase in cell free mtDNA in plasma.
The role of mitochondrial reactive oxygen species in insulin resistance
2022, Free Radical Biology and Medicine
Insulin resistance is one of the earliest pathological features of a suite of diseases including type 2 diabetes collectively referred to as metabolic syndrome. There is a growing body of evidence from both pre-clinical studies and human cohorts indicating that reactive oxygen species, such as the superoxide radical anion and hydrogen peroxide are key players in the development of insulin resistance. Here we review the evidence linking mitochondrial reactive oxygen species generated within mitochondria with insulin resistance in adipose tissue and skeletal muscle, two major insulin sensitive tissues. We outline the relevant mitochondria-derived reactive species, how the mitochondrial redox state is regulated, and methodologies available to measure mitochondrial reactive oxygen species. Importantly, we highlight key experimental issues to be considered when studying the role of mitochondrial reactive oxygen species in insulin resistance. Evaluating the available literature on both mitochondrial reactive oxygen species/redox state and insulin resistance in a variety of biological systems, we conclude that the weight of evidence suggests a likely role for mitochondrial reactive oxygen species in the etiology of insulin resistance in adipose tissue and skeletal muscle. However, major limitations in the methods used to study reactive oxygen species in insulin resistance as well as the lack of data linking mitochondrial reactive oxygen species and cytosolic insulin signaling pathways are significant obstacles in proving the mechanistic link between these two processes. We provide a framework to guide future studies to provide stronger mechanistic information on the link between mitochondrial reactive oxygen species and insulin resistance as understanding the source, localization, nature, and quantity of mitochondrial reactive oxygen species, their targets and downstream signaling pathways may pave the way for important new therapeutic strategies.
Delivery of coenzyme Q10 with mitochondria-targeted nanocarrier attenuates renal ischemia-reperfusion injury in mice
2021, Materials Science and Engineering C
Ischemia-reperfusion (I/R) injury causes high morbidity, mortality, and healthcare costs. I/R induces acute kidney injury through exacerbating the mitochondrial damage and increasing inflammatory and oxidative responses. Here, we developed the mitochondria-targeted nanocarrier to delivery of Coenzyme Q10 (CoQ10) for renal I/R treatment in animal model. The mitochondria-targeted TPP CoQ10 nanoparticles (T-NP_CoQ10) were synthesized through ABC miktoarm polymers method and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The I/R mouse model and oxygen-glucose deprivation/reperfusion (D/R) model were created to examine the role of T-NP_CoQ10 on renal I/R. Mitochondrial DNA damage, apoptosis, and inflammatory cytokines were measured in I/R injury mice. Plasma creatinine, urea nitrogen, tubular injury score was tested to assess the renal function. T-NP_CoQ10 nanoparticles could be delivered to renal mitochondria preciously and efficiently. T-NP_CoQ10 administration attenuated oxidative injury in both cell and animal models significantly, alleviated mtDNA damage, suppressed inflammatory and apoptotic responses, and improved renal function. The mitochondria specific CoQ10 delivery provided a precious and efficient method for protecting inflammatory and oxidative responses of I/R-induced renal damage.

View all citing articles on Scopus

View full text

The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number

Abstract

Section snippets

Theory/rationale

Protocol

Example of derived data

Summary

Acknowledgments

Methods

Mutat. Res.

J. Biol. Chem.

Mutat. Res.

Mutat. Res.

Aquat. Toxicol.

Comp. Biochem. Physiol. C Toxicol. Pharmacol.

Mutat. Res.

Gene

Comp. Biochem. Physiol. C Toxicol. Pharmacol.

Comp. Biochem. Physiol. C Toxicol. Pharmacol.

Aquat. Toxicol.

Nucleic Acids Res.

Genome Biol.

DNA Repair and Mutagenesis

Nat. Rev. Mol. Cell Biol.

PLoS Genet.

Proc. Natl. Acad. Sci. USA