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

Winnie Kam,Connie Banos,Vanessa Lake,Richard Banati,Justin Davies

doi:10.3390/ijms140611544

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.

Highlights

Mitochondria, organelles with a broad range of functions from the provision of cellular energy to signalling, cell cycle and growth, have their own genome consisting of multiple copies of circularDNA [1]
It is composed of four enzymatic complexes, and, together with ATP synthase (Complex V), there are 13 subunits encoded by mitochondrial DNA as well as 77 subunits encoded by nuclear DNA [11]
The present study describes an important limitation in the use of Quantitative polymerase chain reaction (qPCR) for the quantification of both nuclear and mitochondrial gene copy numbers after gamma irradiation, due to radiation-induced fragmentation of the DNA templates

Summary

Introduction

Mitochondria, organelles with a broad range of functions from the provision of cellular energy to signalling, cell cycle and growth, have their own genome consisting of multiple copies of circular. DNA copies have been reported after ionizing irradiation in vivo [2,3,4,5,6] and in vitro [7,8,9] These observations are being interpreted as mitochondrial polyploidization [4], an apparent compensatory effect in response to stress-induced mitochondrial DNA depletion in order to maintain cellular energy status and survival [2,4,10]. The electron transport chain is a system that resides within mitochondria for respiration generation of chemical energy in the form of adenosine triphosphate. It is composed of four enzymatic complexes, and, together with ATP synthase (Complex V), there are 13 subunits encoded by mitochondrial DNA as well as 77 subunits encoded by nuclear DNA [11]. The present study describes an important limitation in the use of qPCR for the quantification of both nuclear and mitochondrial gene copy numbers after gamma irradiation, due to radiation-induced fragmentation of the DNA templates

Results and Discussion

Gel Analysis of DNA Template Fragmentation after Radiation Exposure

Sample Preparation

Gamma Irradiation

DNA Isolation and Gel Electrophoresis

Conclusions