Technical aspects of the electrophoresis stage in the comet assay.

Abstract

Inter- and intralaboratory variability of results is still a serious issue in the comet assay. There are several technical conditions of procedure, which may critically affect the results and electrophoresis terms were identified as main. A comparative assessment of the expected and actual field strength in five electrophoretic tanks and the contribution of the revealed differences to the variability in DNA damage carried out. Only for one tank, the measured field strength coincided with the expected 1 V/cm, while for four it ranged from 0.6 to 2.0 V/cm. The values of DNA damage assessed in the same samples of mouse kidney cells differed between tanks up to 4.7-fold for induced and up to 10-fold for spontaneous DNA damage. High local variations in the field strength and solution temperature across the platform as well as in %DNA in the tail of identical cell samples within electrophoresis runs also revealed. These variations were reduced by recirculation of electrophoresis solution. The results show that discrepancy between the estimated and the actual field strength can be reason of inter-laboratory variation of the comet assay results. Recirculation of the solution during electrophoresis will be useful to control of intra-laboratory and intra-assay variations.