Preservation of comet assay slides: comparison with fresh slides.
The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H(2)O(2)) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22-50 degrees C) and re-hydrated did not differ in the values obtained for H(2)O(2)-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.
Woods
J A
JA
Nutritional Sciences, Department of Food Science and Technology, University College, Cork, Ireland.
O'Leary
K A
KA
McCarthy
R P
RP
O'Brien
N M
NM
D002467:Cell Nucleus / Q000378:metabolism; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000032:analysis; D004249:DNA Damage / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006861:Hydrogen Peroxide; D000432:Methanol; D013048:Specimen Handling; D013696:Temperature; D014021:Tissue Preservation / Q000379:methods*; D014407:Tumor Cells, Cultured
D004247:DNA; D006861:Hydrogen Peroxide; D000432:Methanol