and Wallin,H. . and Marcos,R. Table IV presents the slope significance F-test for each regression line and compares the slopes obtained with and without -ala treatment. A recent review details the many compounds investigated by the Comet assay, which notably include metals, pesticides, opiates, nitrosamines and anticancer drugs (Rojas et al., 1999). The concept underlying the SCGE assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus. Sodium chloride, sodium hydroxide and Triton X-100 were from Merck (Darmstadt, Germany), EDTANa2H2 from Acros Organics (Geel, Belgium) and dimethyl sulphoxide from VWR International (Leuven, Belgium). Singh NP, McCoy MT, Tice RR, Schneider EL. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Basic Clin Pharmacol Toxicol. government site. (1995). We have consequently investigated two often used parameters, the tail DNA and the Olive tail moment, an expression of tail moment little influenced by the difficult to measure tail length parameter. Lee RF, Steinert S. Use of the single cell gel electrophoresis/Comet Assay for detecting DNA damage in aquatic (marine and freshwater) animals. Although image analysis on comets has been preferable for continuity in assessing DNA damage by this method, some groups have been working on simple, less time consuming visual scoring methods that do not require special image analysis software. 2003). Comet assay measures of irinotecan-induced DNA damage in vitro and in vivo. PMC P. Duez and others, Statistics of the Comet assay: a key to discriminate between genotoxic effects, Mutagenesis, Volume 18, Issue 2, March 2003, Pages 159166, https://doi.org/10.1093/mutage/18.2.159. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It combines the simplicity of biochemical techniques for detecting DNA single strand breaks (strand breaks and incomplete . Mutat Res 2003;544:4364. After rehydration (100 l water), staining (200 l ethidium bromide, 20 g/ml, 10 min) and washing, comet-shaped structures were measured by epifluorescence microscopy (Axiovert S100 TV; Zeiss, Jena, Germany) using the Komet 4.0 software (Kinetic Imaging, Liverpool, UK) connected to an Orca-II cooled CCD camera (12 bits, 50C) (Hamamatsu, Japan). Assessing the endocrine disrupting potentials and genotoxicity in environmental samples from Taiwanese rivers. If the DNA migration in control cells does not fall within the laboratorys historical control values, the data generated from that electrophoresis run are rejected. in 1988. Single cell gel electrophoresis, the so-called `Comet' assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. 2000; Hartmann et al. Before As each sample presents essentially similar distributions across electrophoreses, this test was applied, in a first step, to the sample-by-sample comparison of electrophoreses; the probabilities for these KruskalWallis T statistics are presented in Figure 1. The trend analysis appears to be a powerful tool, notably to prove the absence of effect (e.g. There are some commercial sources of control cells such as Trevigen, who prepare their control cells by treating them with known concentrations of etoposide. Green fluorescence was then measured using a circular diaphragm first over the head and then over different positions on the comet tail. 750550, 1,3001,030), so individual pixels cannot be discerned in (a) unless the image is considerably enlarged, Conversion of light intensity to grey values on an image. Given the importance of different measurements in determining the extent of DNA damage (and repair) in this very widely used assay, this article aims to (a) analyse the developments and our current understanding of different Comet Assay measurements (b) analyse their relative importance or use and (c) highlight future developments and perspectives. 2011 Feb;8(2):540-53. doi: 10.3390/ijerph8020540. 2009 Jan-Feb;681(1):3-12. doi: 10.1016/j.mrrev.2008.03.005. This site needs JavaScript to work properly. (, Bauer,E., Reckenagel,R.-D., Fiedler,U., Wollweber,L., Bock,C. Two versions of Comet assay are currently in use; one introduced by Singh et al known as the ''single cell gel electrophoresis (SCGE)'' technique (Singh et al., 1988), in which alkaline electrophoresis is used (pH.13) for analysis of DNA damage and, is capable of detecting alkali labile sites and DNA single-strand breaks in individual . Unauthorized use of these marks is strictly prohibited. (. Genotoxic effect of chronic exposure to DDT on lymphocytes, oral mucosa and breast cells of female rats. Using data from 25 patients, we observed that 2Ds based on tail moment and tail DNA were highly correlated and that tail inertia might provide information on a somewhat different aspect of DNA damage/repair. To confirm whether clouds really represent apoptotic cells, more experiments under different exposure conditions are required bearing in mind that apoptosis is an irreversible process. Other dyes used are SYBR green/gold (Tice et al. Tromethamine (Tris) and -aminolaevulinic acid hydrochloride (-ala) were from Sigma-Aldrich (St Louis, MO). The single cell gel electrophoresis assay (SCGE, also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. The model image is an 8-bit image with available grey values 0255. Subsequently, several research groups published papers in which various Comet Assay parameters were used (Table1). 1988), followed by 4, 6-diamidino-2-phenylindole (DAPI, Gedik et al. This article is published under an open access license. and Hoppe,U. OTM is calculated as a product of two factors: the percentage of DNA in the tail (%Tail DNA) and the distance between the intensity centroids (centres of gravity) of the head and the tail along the x-axis of the comet. et al. Basic Clin Pharmacol Toxicol. Mutagenesis. Ideally, they should be scored visually and recorded alongside the results of image analysis. In the present study, this finding is purely anecdotal as these comparisons make no sense. National Library of Medicine et al., 1994) or measured from morphological parameters obtained by image analysis and integration of intensity profiles (Olive et al., 1990; Kent et al., 1995; Bocker et al., 1997; Olive, 1999). and Magnussen,H. 1. Sham controls were prepared in exactly the same way, but without light exposure. There is a comet in my computer! HHS Vulnerability Disclosure, Help One day before the experiment, cells were harvested by centrifugation (900 g, 2 min) and suspended at a density of 5106 cells in 6 ml of fresh medium in 25 cm2 aerated culture flasks. This phenomenon is particularly seen in treatment with hydrogen peroxide and methyl methanesulphonate (Collins 2004; Speit et al. In addition, the assay is being used in translational research to assess whether tumour radio-sensitivity (Fisher et al. Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available. J Nucleic Acids. An official website of the United States government. sharing sensitive information, make sure youre on a federal and Kupper,L.L. stomach). Comet is a 1985 popular-science book by Carl Sagan and Ann Druyan. The slides are put in lysing buffer to allow the DNA to unwind and then in electrophoresis buffer. The emitted light from individual cells passed an emission filter (long pass 525nm). Dietary Supplementation with the Red Seaweed. TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation. Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, et al. Performance of the comet assay in a high-throughput version. government site. Szeto YT, Benzie IF, Collins AR, Choi SW, Cheng CY, Yow CM, Tse MM. The Comet Assay was first introduced by Ostling and Johanson in 1984. Epub 2009 Feb 11. Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, et al. The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be drawn through an agarose gel in response to an electric field. As OTM values can differ widely between laboratories and/or with different software packages, % Tail DNA is considered appropriate for regulatory or inter-laboratory comparison studies. Such quantification includes both visual examinations (i.e., photographic, occulometer or non-specific image analysis systems) or by use of commercially available or public domain specific image analysis software packages. Google Scholar. When allowed to repair for a period of time, the signals return back to the head. doi:10.1016/j.mad.2011.04.012. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. A trend analysis allowed doseeffect relationships (DNA damage versus dose of light) to be investigated and the curves obtained with and without porphyrin induction to be compared. Dhawan & Anderson (2009): The Comet Assay in Toxicology. The Comet Assay has mainly remained an assay of academic and scientific interest until quite recently. 1988; Comet Assay interest group website: http://cometassay.com/). Bethesda, MD 20894, Web Policies Intracellular DNA damage by lysine-acetylene conjugates. They presented their results in terms of the ratio of fluorescence (Fx) at distance micrometre on the tail versus fluorescence at the centre of the head (Fo). S2CID22466782. This is often regarded as that the original comet assay publication. (1995). One of the major issues is the proper quantification of DNA damage/repair. The stronger the signal from the migrated DNA the more damage there is present. The slides are then immersed in a solution that cause the cells to lyse. Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. This page was last edited on 15 September 2022, at 20:21. Although the 75th percentiles demonstrate a slight similar trend, the slope is not statistically significant, which is probably due to a certain saturation of this statistic at higher break levels. The visible light does not induce any DNA damage in the absence of porphyrins; the determination coefficients and tests for slope significance are largely non-significant (P >> 0.05). in 1990. The exact origin of clouds is not clear, but it is assumed that apoptotic cells lead to clouds. O-hydroxylamine-coupled alkaline gel electrophoresis assay for the detection and measurement of DNA single-strand breaks. Prentice Hall, Upper Saddle River, NJ. Followed by. Mutat Res 2006;605:1721. National Library of Medicine Quartiles with their confidence intervals were computed from each distribution according to Aczel (1993) and Conover (1999) and analysed by ANOVA, t-test or regression analysis with the help of the Systat 7.0 software (Systat Software, http://www.systat.com). et al., 1997; De Boeck et al., 2000) tests; the KolmogorovSmirnov test has also been applied to pooled data from several experiments (Bergqvist et al., 1998), resulting in very high numbers of points which may distort the statistical treatment. Comet Assay or single cell gel electrophoresis (SCGE) is a versatile, simple, and adaptable method to measure DNA damage and repair at individual cell level. They also used it to study SBs rejoining in X-irradiated . and Hellman,B. Such specific software packages also facilitate easy statistical analyses, plotting and documentation of the data. Control cells consistently produce comets with predetermined DNA migration in the tails. Reliable Comet Assay measurements for detecting DNA damage induced by ionising radiation and chemicals. The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals.
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