Simultaneous quantification of DNA damage and mitochondrial copy number by long-run DNA-damage quantification (LORD-Q)

// Benjamin Dannenmann 1 , Simon Lehle 1 , Sebastian Lorscheid 1 , Stephan M. Huber 2 , Frank Essmann 1, * and Klaus Schulze-Osthoff 1, 3, * 1 Department of Molecular Medicine, Interfaculty Institute for Biochemistry, University of Tübingen, 72076 Tübingen, Germany 2 Department of Radiation Oncology, University of Tübingen, 72076 Tübingen, Germany 3 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany * These authors contributed equally to this work Correspondence to: Frank Essmann, email: frank.essmann@uni-tuebingen.de Klaus Schulze-Osthoff, email: kso@uni-tuebingen.de Keywords: DNA damage, genotoxicity, mitochondrial DNA, LORD-Q, qPCR Received: June 19, 2017 Accepted: July 26, 2017 Published: August 10, 2017 ABSTRACT DNA damage and changes in the mitochondrial DNA content have been implicated in ageing and cancer development. To prevent genomic instability and tumorigenesis, cells must maintain the integrity of their nuclear and mitochondrial DNA. Advances in the research of DNA damage protection and genomic stability, however, also depend on the availability of techniques that can reliably quantify alterations of mitochondrial DNA copy numbers and DNA lesions in an accurate high-throughput manner. Unfortunately, no such method has been established yet. Here, we describe the high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) and its suitability to simultaneously measure DNA damage rates and mitochondrial DNA copy numbers in cultured cells and tissue samples. Using the LORD-Q multiplex assay, we exemplarily show that the mitochondrial DNA content does not directly affect DNA damage susceptibility, but influences the efficacy of certain anticancer drugs. Hence, LORD-Q provides a fast and precise method to assess DNA lesions, DNA repair and mtDNA replication as well as their role in a variety of pathological settings.