Comparison of different DNA staining methods in the comet assay

Single-cell gel electrophoresis (SCGE, comet assay) is a sensitive technique for measuring DNA damage at the level of an individual cell (Collins, A.R. 2013). A fluorescent dye commonly used for visualisation of DNA damage is the intercalating agent ethidium bromide. However, ethidium bromide is thought to act as a mutagen and is categorized as carcinogen. The new generation of fluorescent DNA staining agents such as SYBR Green or GelRed are developed to replace highly toxic ethidium bromide. These substances are marketed as less toxic and more sensitive alternatives to ethidium bromide. The aim of our research was to investigate the sensitivity of three different fluorescent dyes, ethidium bromide, SYBR Green I, and GelRed, as well as comparing the obtained results with each other. DNA damage induced by photosensitizer riboflavin (50 µmol/L) in the presence of visible light (Epe et al., 1993) was determined in HT-29 colon carcinoma cells without/ with formamidopyrimidine DNA glycosylase (FPG) treatment. Results show that staining of cells with SYBR Green I and GelRed gave similar tail intensities (TI%) as ethidium bromide. Tail intensities of riboflavin-induced DNA damage without/with FPG treatment were 2.19 / 4.74 TI% for ethidium bromide, 2.17 / 4.82 TI% for SYBR Green I, and 2.12 / 5.05 TI% for GelRed. Similar results were observed for negative control (0.1% DMSO). Taken together, our research demonstrates that all three investigated fluorescent DNA staining agents provide comparable results in HT-29 cells and SYBR Green I as well as GelRed are suitable alternatives to ethidium bromide. To confirm our results a large number of samples should be tested in future.