Electrophoresis in the Comet Assay

The comet assay is a sensitive technique to measure lesions in DNA, based on electropho- retic 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 electropho- resis 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 condi- tions. 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.