Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells

Abstract

In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both the formation of DNA damage as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria or a separate step of mitochondrial DNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol of the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

Fig. 1

(a) Spreadsheet used for DNA quantitation. Example depicts fluorescence values obtained during the first step of quantitation, pre-quantitation, and the graph shows values obtained for the standard curve. Above the graph are all calculations related to the DNA standard curve. The first column represents the concentrations of DNA used as standards. The second and third columns show the raw fluorescence readings. These values were averaged (last column). Below the graph is an example of values obtained for an experimental set of DNA samples. Read 1 and Read 2 columns show raw fluorescence readings for each sample; the third column is the mean of those values. DNA concentration is calculated based on the slope of the standard curve. The last two columns show, respectively, the amount of DNA and of TE buffer necessary to dilute the sample DNA to 3 ng/μL

Fig. 2

Representation of the raw fluorescence values obtained after PCR amplification of the mitochondrial genome of mammalian fibroblasts exposed to 200 μM of hydrogen peroxide for the indicated times. Column one, sample identification; columns two and three, raw fluorescence readings for each sample; fourth column, average of values from first two columns; these values are then background corrected (column 5). Relative amplification (column 6) is calculated comparing the values of the treated samples with undamaged control and is plotted in the left graph. Lesion frequency (column 7) is obtained based on the values plotted on column 6 and are expressed as lesions per 10 kb of the mitochondrial genome (column 8 and right graph)

Fig. 3

PCR products of QIAcube-extracted mouse DNA+/−digestion with HaeII. QIAcube extraction apparently results in mostly covalently closed supercoiled mtDNA, which limits primer access. HaeII digestion near the D-Loop (bp ~2,607) greatly increases amplification of the large target. Raw fluorescence values of Lmito (a) and Smito (b) and hence lesion frequencies (c) are affected by digestion. Lesion frequencies represent the decrease in amplification of the large mitochondrial fragment normalized to the small fragment. Data represent the mean+/−SD of two biological samples. Net fluorescence: Picogreen fluorescence of the PCR product minus a “no-template” control