Electrophoresis in the Comet Assay

The comet assay is a sensitive technique to measure lesions in DNA, based on electrophoretic separation of DNA from cells embedded in agarose. Movement of DNA fragments is determined by the potential (V/cm), the time, and the viscosity of the medium (agarose). There is historically considerable confusion as to other factors, that is, current, liquid depths, circulation of the liquid, and temperature. Lack of standardization of electrophoresis including suboptimal power supplies and electrophoresis tanks causes considerable variations within and between laboratories. Ring trials have not been able to clearly identify the cause(s) of variation. Comparison of comet data from cohorts of human blood lymphocytes is used in the COST project hCOMET to identify early biomarkers of the disease. This calls for standardization of analysis. We performed measurements of electric potentials in a tank using multiple electrodes. Variations (time/position) were reduced by circulating electrophoresis liquid at 10% (volume) per min; this also stabilized the temperature. Circulation was accompanied by only slightly reduced variation in DNA damage among 384 irradiated cell samples electrophoresed concomitantly. In conclusion, comparing data between laboratories and cohorts must give emphasis to electrophoresis conditions. Results should be specified with respect to voltage (V/cm), time, and agarose concentration. We expect that suitable correction factors for these parameters may reduce inter-laboratory variations in comet data, allowing more precise comparison of results from different human cohorts

Towards minimizing variations in the comet assay: The electric potential during electrophoresis and implications of circulating the electrophoresis solution

The comet assay (single cell gel electrophoresis) is a sensitive and versatile technique for measuring DNA damage and repair at the single cell level. Östling and Johanson were the first to introduce the comet assay in 1984, and the method has been continuously improved thereafter. However, considerable variations in results obtained between different laboratories as well as within the same laboratory are still being observed. Even neighboring cell/agarose samples can give highly different levels of DNA damage. Minimizing the variability of the comet assay is highly desirable in order to achieve consistent results in different experiments as well as in collaborative studies involving different laboratories. The assay protocol consists of several steps, one of which involves electrophoretic separation of the negatively charged DNA molecules in an electric field. Variations in the electrophoresis condition, have shown to be a major source of error in the comet assay. Potential difference (V/cm) is the driving force in electrophoresis and control of the local voltages during electrophoresis is thus particularly important in order to reduce variability of the comet assay. The ultimate aim of this thesis was to reduce variation in the comet assay results within and between laboratories, by developing a more stable and robust comet assay protocol. This thesis was chosen to examine systematically the effect of circulating the electrophoresis solution during electrophoresis. Two main experimental works were carried out. The first involved measurements of electric potentials in agarose at defined positions across the platform as a function of electrophoresis time under different experimental conditions. The second included the complete comet assay procedure on human peripheral blood lymphocytes irradiated with X-rays using a high-throughput format with 96 minigels on a plastic support film. No previous studies have investigated in such detail as was done in this thesis regarding the implication of adding circulation of the electrophoresis solution during electrophoresis in the comet assay. This study showed that the variations in the electric potential across the platform were considerably reduced by circulating the electrophoresis solution. It was then anticipated that these variations would be reflected in parallel variations in DNA damage. However, the variations in voltages observed from the electric potential measurements during electrophoresis without circulation, were only partly paralleled with the variations in tail DNA intensities from the comet assay performed with or without circulation of different flow rates during electrophoresis. A decrease of approximately 1-2% in the CV in the per cent tail DNA intensity using the high-throughput format was obtained by circulating the electrophoresis solution at flow rates above 109 ml/min compared to without circulation in this study. In addition, a less variable per cent tail DNA intensity was obtained by circulating the electrophoresis solution. The circulation also contributed to a better temperature stability. Based on these observations, circulation of the electrophoresis solution during electrophoresis in the comet assay is thus recommended. A closer examination of the electrochemical processes occurring at the electrodes was conducted. It was concluded that the electrode system initially constructed for local measurements of potential, were insufficient due to the lack of a reference electrode suitable for the system. A reference electrode must be used in order to determine if the observed variations in electric potentials could have occurred due to minute concentration gradients between the electrodes. Due to limited time available in the thesis this was not accomplished/completed