title: LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis.
abstract: DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.
authors.affiliation: Interfaculty Institute for Biochemistry, Department of Molecular Medicine, University of Tübingen, 72076 Tübingen, Germany, Division of Immunogenetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany, Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Lehle
authors.forename: Dominic G
authors.initials: DG
authors.lastname: Hildebrand
authors.forename: Britta
authors.initials: B
authors.lastname: Merz
authors.forename: Peter N
authors.initials: PN
authors.lastname: Malak
authors.forename: Michael S
authors.initials: MS
authors.lastname: Becker
authors.forename: Peter
authors.initials: P
authors.lastname: Schmezer
authors.forename: Frank
authors.initials: F
authors.lastname: Essmann
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulze-Osthoff
authors.forename: Oliver
authors.initials: O
authors.lastname: Rothfuss
mesh_terms: D002467:Cell Nucleus / Q000235:genetics*; D002478:Cells, Cultured; D020552:Comet Assay; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000737:chemistry; D054629:Genome, Mitochondrial; D006801:Humans; D019169:Jurkat Cells; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial