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Analysis of Oxidative Damage by Gene‐Specific Quantitative PCR

Olga A. Kovalenko¹, Janine H. Santos¹

¹University of Medicine and Dentistry of New Jersey, Newark, New Jersey

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 19.1

DOI:

10.1002/0471142905.hg1901s62

Online Posting Date:

July, 2009

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Abstract

This unit describes the gene-specific quantitative PCR-based (QPCR) assay, which is used to measure DNA integrity of both nuclear and mitochondrial genomes based on amplification of long DNA targets. QPCR can be used to quantify the formation of DNA damage and the kinetics of DNA repair by following restoration of amplification of the target DNA over time after removal of the damaging agent. A detailed protocol to set up QPCR in any laboratory, highlighting critical parameters for successful establishment of the assay and interpretation of the results, is provided here. Advantages (e.g., the use of nanogram amounts of DNA) and limitations (e.g., the inability to define the specific type of lesion present on the DNA) of using QPCR to assay DNA damage in human cells are also described. Curr. Protoc. Hum. Genet. 62:19.1.1-19.1.13. © 2009 by John Wiley & Sons, Inc.

Keywords: mitochondrial and nuclear DNA integrity; oxidative stress; reactive oxygen species; DNA damage and repair; QPCR

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Introduction
Basic Protocol 1: QPCR for Nuclear and Mitochondrial DNA Integrity
Basic Protocol 2: Estimation of DNA Damage
Support Protocol: Sample Preparation and DNA Quantification
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: QPCR for Nuclear and Mitochondrial DNA Integrity

Materials

Sample genomic DNA (see Support Protocol)
GeneAmp XL PCR kit (Applied Biosystems cat. no. N808-0188), including:
- rTth DNA polymerase XL (2 U/µl)
- 3.3× XL PCR buffer
1 mg/ml bovine serum albumin (BSA, Roche cat. no. 10711454001)
dNTPs: 10 mM of total solution (2.5 mM of each nucleotide, GE cat. no. 27-2035-01) diluted in water, in 100-µl aliquots, and frozen at –80°C
Primers (see Table 19.1.1 and Critical Parameters): dilute in water for a working concentration of 10 µM
Magnesium (Mg²⁺)

Dedicated PCR hood for setting up reactions, e.g., Purifier PCR Enclosure (Labconco cat. no. LC-LO-3970302) or a tissue culture hood
200-µl PCR tubes
Thermal cyclers (e.g., Biometra)

Basic Protocol 2: Estimation of DNA Damage

Materials

20× TE buffer: 200 mM Tris×Cl, 20 mM EDTA, pH 7.5
PCR product samples and blank (see Basic Protocol 1)
5 µl/ml Quant-iT PicoGreen dsDNA reagent (Molecular Probes, Invitrogen cat. no. P7581) in 1× TE buffer

96-well plates
Aluminum foil
Plate reader (e.g., Victor³ Multilabel reader, PerkinElmer) with excitation wavelength at 485 nm and emission wavelength at 535 nm
Data collection software, e.g., Excel

Support Protocol: Sample Preparation and DNA Quantification

Materials

Cell culture or tissue
Genomic DNA buffer set (Qiagen cat. no. 19060)
20× TE buffer: 200 mM Tris×Cl, 20 mM EDTA, pH 7.5
Lambda ()/HindIII DNA (GIBCO cat. no. 15612-013) diluted in 1× TE buffer to make the following 20, 10, 5, 2.5, and 1.25 ng/µl DNA standards
5 µl/ml Quant-iT PicoGreen dsDNA reagent (Molecular Probes, Invitrogen cat. no. P7581) in 1× TE buffer

Genomic-tips (Qiagen cat. no. 10223)
50°C water bath
96-well plates
Aluminum foil
Plate reader (e.g., Victor³ Multilabel reader, PerkinElmer) with excitation wavelength at 485 nm and emission wavelength at 535 nm
Data collection software, e.g., Excel

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Figures

Figure 19.1.1

Cycle parameters utilized for amplification of human targets by QPCR. Two PCR profiles (A and B) with different parameters (see Table 19.1.2).

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Figure 19.1.2

Quantitation of mtDNA damage and repair kinetics. A shows fluorescence values of mitochondrial genome amplification products for normal human fibroblasts treated with 200 µM H₂O₂ for 1 hr and allowed to recover for 0, 6, 12, and 24 hr after treatment. The first column shows designated samples, the second and third columns show raw fluorescence values for each sample, the forth column shows the average of the two previous column values, and the fifth column shows background subtracted from the average values in the previous column. Column 6 represents a calculation of relative amplification of the treated samples compared to untreated control. B shows the values from column 6. Lesions in column 7 are calculated based on final readings values from column 6, according to the formula ln(A_T/A_C). Column 8 and C represent number of lesions per 10-kb of the mitochondrial genome.

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Figure 19.1.3

DNA quantitation in an Excel spreadsheet. A represents calculation of the DNA standard curve. The first column contains DNA standard concentrations, the second column represents final values for the fluorescent readings (with background fluorescence subtracted for each standard concentration). Values in the third and fourth columns are raw fluorescent readings for each standard, with averages of these values shown in the last column. B is an example of DNA quantitation for experimental samples. The first and second columns are raw fluorescence readings for the samples; the third column is the mean of those values. “Net” is the average value of the readings with background fluorescence subtracted. The slope of the standard curve is calculated and DNA concentration is calculated based on the slope. The last two columns show calculations for the amount of DNA and of 1× TE buffer necessary to prepare the sample at 10 ng/µl DNA concentration.

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Literature Cited

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