Comparison of different DNA staining methods in the comet assay
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University of Kaiserslautern, Department of Chemistry, Division of Food Chemistry & Toxicology, Germany
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.
References
References:
Collins, A. R. (2013). Measuring oxidative damage to DNA and its repair with the comet assay. Biochimica Biophysica Acta,13, 4158–4165.
Epe, B., Pflaum, M., Boiteux, S. (1993). DNA damage induced by photosensitizers in cellular and cell-free systems. Mutation Research, 299, 135–145.
Keywords:
DNA staining agents,
Comet Assay,
ethidium bromide,
GYBR Green I,
Gelred
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Poster Presentation
Topic:
New applications and technical improvements
Citation:
Bakuradze
T and
Richling
E
(2015). Comparison of different DNA staining methods in the comet assay.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00007
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Received:
02 Jun 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
Prof. Elke Richling, University of Kaiserslautern, Department of Chemistry, Division of Food Chemistry & Toxicology, Kaiserslautern, 67663, Germany, richling@chemie.uni-kl.de