Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH>13)?
The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt-detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH>13 [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184-191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205-214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt-detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH>13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3M NaOH, 0.02 M Trizma and 1mM EDTA, pH>13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes.
Vivek Kumar
P R
PR
Low Level Radiation Research Laboratory, Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Kollam 691 001, Kerala, India.
Cheriyan
V D
VD
Seshadri
M
M
D000468:Alkalies / Q000494:pharmacology*; D020552:Comet Assay / Q000379:methods*; D006801:Humans; D008297:Male; D012972:Sodium Hydroxide / Q000494:pharmacology
D000468:Alkalies; D012972:Sodium Hydroxide