The influence of temperature during alkaline treatment and electrophoresis on results obtained with the comet assay.
The alkaline comet assay (single-cell gel electrophoresis) is becoming established as a genotoxicity test with many fold applications in vitro and in vivo. While the underlying principles are identical, various modifications of the method are in use which clearly affect the sensitivity and resolving power of the assay. One variable of potential importance that has been disregarded until now is temperature during alkaline treatment and electrophoresis. We therefore performed comet assay experiments with human blood and V79 Chinese hamster cells using two different temperatures (4 and 20 degrees C, i.e. room temperature) during alkaline treatment and electrophoresis. DNA damage was induced by the two standard mutagens gamma irradiation and methyl methanesulfonate (MMS). The results clearly indicate significant differences in the detection of background and mutagen-induced DNA damage at these two temperatures. Under otherwise identical test conditions (including the duration of alkaline treatment and electrophoresis), increased temperature during alkaline treatment and electrophoresis strongly enhances DNA migration. Our findings suggest that the comet assay should be performed under strictly controlled and reproducible temperature conditions. In any case the temperature during alkaline treatment and electrophoresis should be stated in a publication to allow for a critical evaluation of results obtained with the comet assay.
Speit
G
G
Universitätsklinikum Ulm, Abteilung Medizinische Genetik, Germany. guenter.speit@medizin.uni-ulm.de
Trenz
K
K
Schütz
P
P
Rothfuss
A
A
Merk
O
O
D000468:Alkalies; D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D004587:Electrophoresis, Agar Gel; D005720:Gamma Rays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity; D013696:Temperature
D000468:Alkalies; D000477:Alkylating Agents; D009153:Mutagens; D004247:DNA; D008741:Methyl Methanesulfonate