title: Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.
abstract: Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.
authors.affiliation: Australian Nuclear Science and Technology Organisation, Lucas Heights, Sydney, New South Wales 2234, Australia. wik@ansto.gov.au.
authors.forename: Winnie W Y
authors.initials: WW
authors.lastname: Kam
authors.forename: Vanessa
authors.initials: V
authors.lastname: Lake
authors.forename: Connie
authors.initials: C
authors.lastname: Banos
authors.forename: Justin
authors.initials: J
authors.lastname: Davies
authors.forename: Richard
authors.initials: R
authors.lastname: Banati
mesh_terms: D002467:Cell Nucleus / Q000235:genetics*; D003080:Cold Temperature; D053938:DNA Fragmentation / Q000528:radiation effects; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000528:radiation effects; D004587:Electrophoresis, Agar Gel; D005720:Gamma Rays; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000235:genetics*; D011123:Polyploidy; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
chemical_list: D004272:DNA, Mitochondrial