Analysis of oxidative damage by gene-specific quantitative PCR.
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.
Kovalenko
Olga A
OA
University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA.
Santos
Janine H
JH
D000818:Animals; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D005796:Genes; D016678:Genome; D006801:Humans; D018384:Oxidative Stress / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017382:Reactive Oxygen Species / Q000378:metabolism
D017382:Reactive Oxygen Species; D004247:DNA