A duplex real-time PCR assay for detection of drug-induced mitochondrial DNA depletion in HepG2 cells.
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.
Höschele
Doris
D
Federal Institute for Drugs and Medical Devices, D-53175 Bonn, Germany. hoeschel@bfarm.de
Wiertz
Martina
M
Garcia Moreno
Inmaculada
I
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000032:analysis / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D006801:Humans; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D004272:DNA, Mitochondrial