Detection and quantification of a radiation-associated mitochondrial DNA deletion by a nested real-time PCR in human peripheral lymphocytes

Abstract

In this study we implemented a new assay using a nested real-time polymerase chain reaction (PCR) to detect radiation-induced common deletion (CD) in mitochondrial DNA (mtDNA) of human peripheral lymphocytes. A standard curve for real-time PCR was established by applying a plasmid DNA containing human normal mtDNA or mutated mtDNA. Human peripheral lymphocyte DNA was amplified and quantified by real-time PCR using primer sets for total damaged or mutated mtDNA, plus probes labeled with the fluorescent dyes. The first-round PCR generated multiple products were used as the template for a second-round PCR. We herein describe a nested real-time PCR assay capable of quantifying mtDNA bearing the CD in human peripheral lymphocytes following exposure (in vitro) to 137Cs γ-rays in a dose range of 0.5 up to 5Gy. The reproducibility of this assay was evident for both unirradiated and irradiated samples by examining human blood lymphocytes from 14 donors. This technique was sensitive enough to detect deletions in mtDNA at low dose levels, as low as 0.5Gy, and higher levels of CD mtDNA were evident at higher doses (≥1Gy), however, there was no consistent dose–response relationship.