Comet assay: rapid processing of multiple samples.
The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.
McNamee
J P
JP
Health Canada, Radiation Protection Bureau, 775 Brookfield Road, Postal Locator 6303B, Ottawa, Ontario, Canada. james_mcnamee@hc-sc.gc.ca
McLean
J R
JR
Ferrarotto
C L
CL
Bellier
P V
PV
D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes; D006801:Humans; D006861:Hydrogen Peroxide / Q000009:adverse effects; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D011419:Propidium; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes; D011419:Propidium; D004247:DNA; D006861:Hydrogen Peroxide