Standardization of the comet assay technique on FRTL5 cells.
The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. The principle of break detection, using either the alkaline or neutral version of the assay, makes it a good technique for studying both double and single strand DNA breaks. Furthermore, the possibility of following DNA damage at different time moments also makes it possible to investigate the cell repair mechanisms. This explains why in the last few years there has been a tremendous increase in the number of laboratories which started to use this technique. The technique was first created for lymphocyte cells and later on has been used on many other cell types, growing both in suspension and adherent. To date, no one has applied this technique on normal differentiated endocrine cells, such as FRTL5 cells (Fisher Rat Thyroid Cells). The aim of this study has been to standardize the alkaline version of the Comet Assay technique on FRTL5 cells by studying the kinetics of DNA-damage and DNA-repair after different doses of UV-C (254 nm). FRTL-5 cells not only resulted very sensitive to UV-C (p<0.05 at 5 J/m2), but were also able to repair most of their DNA damage very rapidly (within one hour) as shown by a significant exponential regression in comet length. Finally, the successful measurement of biomarkers of UV-C on thyroid cells established the comet assay as a valuable tool in measurement of DNA damage and repair. Any radiation, or other damaging agents, interacting with living organisms could cause DNA damages which, depending upon dosages and kinetics of exposure, may or may not be completely repaired.
Francesconi
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Dipartimento di Patologia e Medicina Sperimentale e Clinica, Universita di Udine, Udine, Italy.
Del Terra
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E
Meli
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Ambesi-Impiombato
F S
FS
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured / Q000528:radiation effects; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000166:cytology*; D014466:Ultraviolet Rays