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A duplex real-time PCR assay for detection of drug-induced mitochondrial DNA depletion in HepG2 cells.

Research paper by Doris D Höschele, Martina M Wiertz, Inmaculada I Garcia Moreno

Indexed on: 11 Jun '08Published on: 11 Jun '08Published in: Analytical Biochemistry: Methods in the Biological Sciences



Abstract

Screening of drug-induced mitochondrial DNA (mtDNA) depletion during early preclinical drug development is of major interest. Here we describe the establishment of a novel duplex calibrator-normalized real-time polymerase chain reaction (PCR) assay for rapid and reliable quantification of mtDNA in HepG2 cells. This assay involves quantification of an mtDNA target gene (cytochrome b) relative to a nuclear DNA (nDNA) reference gene (beta-actin) in one tube. The assay was evaluated for its precision, linearity, and reproducibility, and reliable detection of mtDNA depletion was demonstrated. Using this novel real-time PCR assay, drug-induced mtDNA depletion could be accurately detected.