Modified Comet Assay Detecting DNA Injury of Whole Blood Lymphncyte Resulted from Ultroviolet Radiation

Objectives To identify the best sample processing of comet assay through different methods of lymphocyte extraction from whole blood and to compare the influence of different layers of gel on percentage of comet cell after whole blood ultraviolet radiation.Methods The slides layered with cell and gel were lysed in a cold lysing solution of pH 10 for 1h.Pre electrophoresis was performed in alkaline electrophoresis runing buffer for 20 min, electrophoresised at 25V 30mA for 20 min.Then neutralized in neutralization for 15 min for twice.Lymphocyte neuclei were stainedwith 5μg/ml PI for 15min.The percentage of comet cells was counted.The ultroviolet radiation in vitro inducing DNA damage in human lymphocyte was measured with comet assay established by our lab.Results Lymphocyte extraction in saline and PBS buffer under ice bath respectively induced the same percentage of comet cell compared with whole blood.But the DNA damage increased when extracting the lymphocyte under room temperature.The percentage of comet cell of whole blood radiated by ultroviolet for 30min and 45min were higher than the unradiated blood, and difference was significant(P0 05) .The difference between one layer and two layers gel was not significant(P0 05).Conclusion It is simple and saving time to take blood as sample to detect lymphocyte DNA damage, and the result was reliable.There was no DNA damage caused by blood processing .Ultraviolet radiation can induce DNA strand breakage in a dose and time dependent manner.To decrease the layers of gel can save a lot of time, and the result had no difference compared with two layers gel.