Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells.
BACKGROUND
The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility.


METHODS
Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique.


RESULTS
In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays.


CONCLUSIONS
This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.
Bauch
T
T
Institut für Medizinische Strahlenbiologie, Universitätsklinikum Essen, Germany.
Böcker
W
W
Mallek
U
U
Müller
W U
WU
Streffer
C
C
D000328:Adult; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000528:radiation effects*