Assesing DNA damage induced by ionising radiation using comet assay

Abstract

Ionizing radiation can cause different kinds of DNA damage - single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. Comet assay (an alkaline version of the single cell gel electrophoresis assay) lets measure DNA damage at the level of individual cell and in this study was used to evaluate CHO cell line DNA damage induced by ionising radiation. CHO cells were irradiated with different doses (2-10Gy) of 6MV energy x-ray radiation, within a 2 hour span embedded in agarose slides, lysed, in alkaline solution exposed to an electric field(~0.74 V/cm, 300mA), painted with ethidium bromide and visualized using fluorescence microscope. In parallel with the comet assay clonogenic cell viability assay was performed. During electrophoresis damaged DNA extends towards anode and form so called comet tail. The amount of DNA migrated outside of cell boundaries to agarose is proportional to the DNA damage caused by ionising radiation. DNA damage was evaluated using open source OpenComet software by measuring the relative intensity of comet head and tail fluorescence. Comet assay results show a linear relationship between radiation dose and DNA damage. This relationship agrees with other studies[1]. After cell viability assay was performed cell survival curve showed higher cell viability compared with a typical cell survival curve[2], dose was ~40% lower. After closer inspection of irradiation setup it was deduced that the air cavity in the Petri dish caused sharp drop in the dose distribution, therefore cell were irradiated with lower dose.