Ethidium bromide (EB) is most commonly used to stain the DNA on Comet Assay slides (Singh et al. PMC Unlike the SYBR green stain, EB does not fade during the process of image capturing. Moreover, visual scoring has the potential to be used for inter-laboratory comparisons. The slides are then immersed in a solution that cause the cells to lyse. (, Moretti,M., Villarini,M., Scassellati-Sforzolini,G., Santroni,A.M., Fedeli,D. -ala photodynamic treatment with visible light irradiation). Would you like email updates of new search results? Comet assay: A brief history | SpringerLink The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. 2023 Mar;18(3):929-989. doi: 10.1038/s41596-022-00754-y. Fluorescence in situ hybridization with comets. 2019 Dec 30;41:24. doi: 10.1186/s41021-019-0140-9. Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. The tail moment calculated by Olive et al. ,Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor, Last edited on 15 September 2022, at 20:21, Learn how and when to remove this template message, "A simple technique for quantitation of low levels of DNA damage in individual cells", "Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo Genetic Toxicology Testing", 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J, "Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay)", "The comet assay for DNA damage and repair: principles, applications, and limitations", "The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures", "DNA damage and repair following treatment with ionizing radiation", https://www2.le.ac.uk/departments/csmm//SCG%20Electrophoresis.pdf, "Clinical significance of sperm DNA damage in assisted reproduction outcome", "The effects of ascorbate and @-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa", "Emerging applications of the single cell gel electrophoresis (Comet) assay. In all our studies (up to stress levels leading to a median 70% tail DNA), both tail DNA and Olive tail moment essentially reach the same conclusions and no obvious differences have been seen between the two parameters with regard to the investigated statistics. The dispersed data obtained in the Comet assay can be efficiently analysed by the proposed statistical treatments, leading to reliable conclusions on observed trends. electrophoresis time), and algorithms used to define the CG of DNA distribution vary among different software packages. MeSH (1995). After a 2226 h stay in the CO2 incubator, 100 l of a fresh solution of -ala was added up to 300 M and the flask was maintained in the incubator for 3 h. The stopper was hermetically closed and the flask exposed for 560 min to the light from either a typical UVA tanning lamp (total output of an array of four tanning tubes, average fluence rate at the cell level 0.31 mW/cm2; Philips Cleo 15 W) or visible light (total output of three spots, average fluence rate at the cell level 11.9 mW/cm2; Philips Spotone R95, 75 W). Kiwifruit protects against oxidative DNA damage in human cells and in vitro. To whom correspondence should be addressed at: Tel: +32 2 650 5242; Fax: +32 2 650 5187; Email, pduez@ulb.ac.be. Methods derived from the general linear model, including the ANOVA, can then be applied to estimate the uncertainties associated with the different sources of variability (Lovell et al., 1999). (1997a) showed that the results of visual scoring correlated very well with image analysis measurements. 2007), and there is a move to replace some traditional assays (e.g. 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. Silver-stained comet assay for detection of apoptosis. This site needs JavaScript to work properly. head and tail). After overnight lysis at 4C in a mixture of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.0:DMSO:Triton X-100 (89:10:1), the slides were washed twice with the electrophoresis buffer (300 mM NaOH, 1 mM EDTANa2H2, pH > 13), incubated for 40 min and electrophoresed (18C, 20 min, 0.7 V/cm, 300 mA, 24 slides/run) in a Gibco Horizon 20-25 tank (Invitrogen) connected to a Power Pac 200 unit (Bio-Rad, Hecules, CA). Data from three different electrophoresis runs are presented, treatment by treatment. SYBR gold stains both double-stranded and single-stranded DNA and is considered better than SYBR green. (, Olive,P.L., Banath,J.P. Clipboard, Search History, and several other advanced features are temporarily unavailable. (, Olive,P.L., Johnston,P.J., Banath,J.P. Kleinbaum,D.G. Toxicol Lett 2004;146:1518. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell. PMID21600238. and Steel,G.G. Please enable it to take advantage of the complete set of features! These tests are clearly overly sensitive, which is most probably due to the high number of comets compared (n100). (1995). This would allow clinicians to individualise patient management, allocating cancer therapy to those for whom it will be of most benefit and reducing the likelihood of patients receiving toxic (and as such ineffective) therapy. endonucleases) increase DNA migration, whereas DNADNA and DNAprotein cross-links result in retarded DNA migration compared to those in concurrent controls (Tice et al. Acta Biochim Pol 2001a;48:6114. Nutr Cancer 2001b;39:14853. It is interesting to note that visual scoring based on Collins et al. Google Scholar. 8). and Olive,P. On the other hand, the proposed statistics may not be appropriate to the data distribution patterns; parametric tests indeed assume Gaussian distributions, whereas non-parametric tests require essentially similar distribution curve shapes (Zar, 1996; Conover, 1999; Statsoft Inc., 1999). (, Bocker,W., Bauch,T., Muller,W.U. Our attempts to estimate the within-electrophoresis reproducibility (MannWhitney test for each pair of duplicate slides) met with the same problem. If the light emitted by the comets is very intense, the image analysis software cannot accurately define certain Comet Assay measurements like OTM, because the centre of gravity (CG) of DNA distribution is not defined correctly (Fig. Disclaimer. During irradiation, the flask temperature was regulated at 4C with the help of a circulating water bath. Google Scholar. B Optimal concentration of DNA binding dye is used and the center of gravity of DNA distribution is properly defined. Introduction The Comet Assay or single cell gel electrophoresis(SCGE) assay is a rapid, sensitive and relatively simplemethod for detecting DNA damage at the level ofindividual cells (Singh et al.1988; Comet Assay in-terest group website: http://cometassay.com/). 1988; Comet Assay interest group website: http://cometassay.com/). Careers. McKenna DJ, Rajab NF, McKeown SR, McKerr G, McKelvey-Martin VJ. (1995) published a visual scoring method that classifies comets from grades 04 (Fig. [14] The most common form of the assay is the alkaline version although there is as yet no definitive alkaline assay protocol. Based on our current understanding however, as mentioned later, this measurement is not considered to be robust. Miyamae Y, Iwasaki K, Kinae N, Tsuda S, Murakami M, Tanaka M, et al. The present work investigates whether these conditions are verified in the Comet assay, using data obtained on a murine leukaemia cell line (P388D1) photodynamically stressed after porphyrin induction. (PDF) Genotoxicity of Contaminants: Comet Assay - ResearchGate Several reviews have been published in recent years to highlight the procedures, advantages and limitations of this assay in genotoxicological, ecotoxicological and biomonitoring studies (Collins 2004; Dixon et al. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. They concluded that only a few comet measurements provided by the image analysis software correlated well with gamma radiation dose. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Dixon DR, Pruski AM, Dixon LRJ, Jha AN. From the present study, we can conclude that a number of the proposed approaches may not be entirely valid. in 1988. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet. 2002; Fairbairn et al. Supermen. Food Chem Toxicol 2007. and transmitted securely. A close examination of this figure shows that whenever this test demonstrates significant variations between electrophoreses (P < 0.05), these differences are objectively insignificant when compared with the differences between treatments. 2007). 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. All the other treatments differ from the controls and from each other, except for the pairs UVA 30 min versus -ala + visible light 30 min and UVA 30 min versus -ala + visible light 60 min; these latter two comparisons depend on the quartile and comet parameter under consideration, which reflects very similar comet distributions (Figure 1). The model image is an 8-bit image with available grey values 0255. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. By October 2005 the publication has been cited 1650 times (Web of Science). About 70 biomonitoring studies have reported DNA damage using visual scoring criteria (Moller 2006). University of Ljubljana, http://www.botanika.biologija.org/exp/comet/Comet-principles.pdf (accessed 07, 2007); 2004. Tail length increases with damage initially and then reaches a maximum Advantage of Comet Assay 1. Biochem Biophys Res Commun 1984;123:2918. Available at http://www.statsoft.com/textbook/stathome.html. Exp Cell Res 1997;232:40711. Clouds are therefore scored only by visual analysis. It was first published in paperback by Signet/New American Library in February 1986. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). There was no recommendation for use of control cells in any of the IWGT workshops. The cells are embedded in agarose and lysed, leaving unbroken DNA in a supercoiled state; strand breaks relaxing the supercoiling are revealed on electrophoresis, free loops of DNA extending to the anode to form a comet-shaped structure. Kruman II, Kumaravel TS, Lohani A, Pedersen WA, Cutler RG, Kruman Y, et al. and Browe,M.A. Tice RR, Furedi-Machacek M, Satterfield D, Udumudi A, Vasquez M, Dunnick JK. 8600 Rockville Pike Comet Assay: Full Protocol to Assess DNA Damage in Cells - ResearchTweet Epub 2008 Mar 30. micronucleus assay) whilst determining the genotoxicity (Raisuddin and Jha 2004). New measure of DNA repair in the single-cell gel electrophoresis (comet 2. (1988) developed the alkaline version of the Comet Assay in which they used the length of DNA migration (tail length) to quantify the extent of damage. PubMed (1988) published the first work using the alkaline comet assay, where the DNA helix is unwound by alkaline treatment, and as a consequence of which ALS are converted to breaks. 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. A comet assay can determine the degree of DNA fragmentation in sperm cells. Mol Biotechnol 2004;26:24961. Good scientific judgement should be used in interpreting these data, and further work is necessary to assess how to integrate clouds with other Comet Assay measurements. Mutat Res 2006;605:716. In the reproducibility study, nine cell treatments (two controls and seven different photodynamic stresses) were performed on independent samples and analysed in triplicate electrophoresis runs, two slides replicating each treatment in each electrophoresis. Yang WY, Cao Q, Callahan C, Galvis C, Sang QX, Alabugin IV. A glass cover slip is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. (, Holz,O., Jorres,R., Kastner,A., Krause,T. Before The first group performed electrophoresis under highly alkaline conditions (pH > 13). and Falcioni,G. Genotoxicity of environmental agents assessed by the alkaline comet assay. The computation procedure includes the application of linear regression. Bethesda, MD 20894, Web Policies Although the tail DNA has been reported as the best comet parameter, increasing linearly with stress intensity up to 75% (Collins et al., 1996), the tail moments are designed to take into account differences in DNA migration that may be due to the nature and extent of DNA relaxation (Olive et al., 1990; Hellman et al., 1995; Kent et al., 1995). Statistics of tail DNA distributions as a function of light dose; each distribution corresponds to 100 comets measured on two slides. and Kirsch-Volders,M. (B) Data for Olive tail moment. 3), but they did not calculate the total score for each gel. 2000;35(3):206-21. doi: 10.1002/(sici)1098-2280(2000)35:3<206::aid-em8>3.0.co;2-j. The slides are then neutralised to pH 7, stained with a DNA-specific fluorescent stain and analysed using a microscope with an attached CCD (charge-coupled device essentially a digital camera) that is connected to a computer with image analysis software. and transmitted securely. Slider with three articles shown per slide. O-hydroxylamine-coupled alkaline gel electrophoresis assay for the detection and measurement of DNA single-strand breaks. Irwin, Burr Ridge, IL, USA. in 1988 and Olive et al. However, for peer-review publications, normally, only one set of measurements (e. g. either tail moment or % Tail DNA) is presented. These statistics notably present a low sensitivity to the few extreme values that can be encountered in comet populations (the so-called apoptotic cells). In order to investigate the doseeffect relationships DNA damage versus dose of light, the median and 75th percentile of tail DNA were computed for each comet distribution and plotted as a function of light dose for both UVA and visible light (Figure 2). ), which recognise oxidised purines and pyrimidine bases, this assay has been used to determine oxidative DNA damage that has been implicated in several health conditions (Collins et al. (, Singh,N.P., McCoy,M.T., Tice,R.R. [18] [19] [20] [21] However, various phenolic compounds are known for their anticancer property. MMS Commun 1995;3:10315. 1. (1995) and Collins et al. Epub 2008 Mar 13. Olive PL, Banath JP, Durand RE. The damage usually detected are single strand breaks and double strand breaks. The exact origin of clouds is not clear, but it is assumed that apoptotic cells lead to clouds. This mono-suspension is cast on a microscope slide. Bethesda, MD 20894, Web Policies The site is secure. However this is not true, both single- and double strand breaks are also detected in neutral conditions. Environ Mol Mutagen 1997a;30:13946. Comet Assay - an overview | ScienceDirect Topics 2003; Kumaravel and Jha 2006). There are still several issues to be resolved before the comet assay is accepted as a standard assay for detecting DNA damage and repair in a single cell. Federal government websites often end in .gov or .mil. For calibration, hydrogen peroxide is usually used to provide a standardized level of DNA damage. and Sarasin,A. The overall structure resembles a comet (hence "comet assay") with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. HHS Vulnerability Disclosure, Help -Number of cells in each comet assay class. | Download Table - ResearchGate Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (Comet) assay. It is generally accepted that visual scoring is as comparable as image analysis; however, image analysis can provide additional information (e.g. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. During electrophoresis the broken DNA moves towards the anode forming a Comet tail, with the greater the extent of damage, the greater the tail. A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies. stomach). The next step is to define the head and the tail of the comet. P1-0212). The normality of distributions was visually appreciated from normal probability plots and objectively evaluated by the ShapiroWilk W-test (Shapiro and Wilk, 1965) using the Analyse-it software (Analyse-It Software Ltd, Leeds, UK, http://www.analyse-it.com). and Hoppe,U. Radiat Oncol. Seidel C, Lautenschlger C, Dunst J, Mller AC. PMC (1990), who used the concept of the tail moment to describe DNA migration. 1997a; Dixon et al. Such classical non-parametric approaches are then unlikely to yield any useful information in comparing treatments and should certainly not be applied to demonstrate a genotoxic effect. [17][18], The comet has been modified for use with sperm cells as a tool for male infertility diagnosis [19][20][21], To break down these tightly bound protamine proteins in order to use the comet for sperm, additional steps in the de-condensation protocol are required. 2012;920:307-13. doi: 10.1007/978-1-61779-998-3_21. Article J Neurosci 2002;22:175262. (, Bock,C., Dittmar,H., Gemeinhardt,H., Bauer,E. Comet Assay measurements: a perspective | SpringerLink At the IWGTP, however, the expert panel did not recommend any particular measurement of comet migration to be more useful than any other measure. Ostling O, Johanson KJ. 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). CAS The authors approached this question by performing alkaline Comet Assay on human peripheral blood lymphocytes irradiated with graded doses of 137Cs gamma radiation and correlating the various comet measurements with the radiation dose. This data is usually presented along with other Comet Assay measurements and cytotoxicity data where an increase in clouds parallels an increase in DNA migration, but this is not always the case. Single cell gel/comet assay: guidelines for in vitro and in vivo In an attempt to make the assay more sensitive and reliable, several research groups have come out with unique procedures and specialised measures of DNA migration. These observations suggest that, in addition to apoptosis, clouds are also induced by high levels of DNA damage as well as in necrotic cells. The more versatile alkaline method of the comet assay was developed by N.P. Immediately after irradiation, the cells were isolated (centrifugation at 900 g, 2 min), washed and resuspended in cold PBS at a density of 2.5106 cells/ml. The extent of DNA migration depends directly on the DNA damage present in the cells. 2003). This site needs JavaScript to work properly. The pixel column number is indicated in italics. in 1988. and Wallin,H. 2008 May;23(3):207-21. doi: 10.1093/mutage/gen014. Singh NP, Stephens RE, Schneider EL. The story Comet- I begin with a moonless night in December in which a cool breeze was flowing. The relationship between the grey value of a pixel on the image and light intensity (fluorescence) that a camera element detects is linear (c), Segmentation of the comet image. Comet Assay - an overview | ScienceDirect Topics Collins BH, Horsk A, Hotten PM, Riddoch C, Collins AR. Raisuddin S, Jha AN. Bookshelf Please enable it to take advantage of the complete set of features! Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. Vol. in 1990. Singh and co workers in 1988. Subsequently, several research groups published papers in which various Comet Assay parameters were used (Table1). Mutat Res 2006;605:1721. the number of pixels per micrometre is known for different microscope objectives). The slides were immersed in two lysis solutions for 30 min each at room temperature. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). 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. Part of Springer Nature. 1988), followed by 4, 6-diamidino-2-phenylindole (DAPI, Gedik et al. As DNA damage produced is directly proportional to the radiation dose, any change in dose should be reflected in proportional change in the comet measurements. (, Moller,P., Knudsen,L.E., Loft,S. Clouds or hedgehogs are important observations in most Comet Assay experiments. Permissions team. J Nucleic Acids. Robust Comet Assay data and interpretation depend on good and optimum slide staining, adoption of robust image analysis practices and use of reliable and meaningful Comet Assay measurement (e.g. Mutagenesis 2003;18:4551. Federal government websites often end in .gov or .mil. (A) Data for tail DNA. 2003) as well as of possible diagnostic use (Kumaravel and Bristow 2005). There have, however, been significant modifications, and the reader is referred to the detailed protocol (currently in use in our laboratory) that was recently published (Collins and Azqueta 2012a ). They also used it to study SBs rejoining in X-irradiated . It is based on nuclear decompaction followed by electrophoresis and visualization of individual spermatozoa. Before it can be accepted as a regulatory tool, this assay has to be harmonised in terms of its methodology and interpretations and should be demonstrated to be reliable, accurate and transferable between laboratories. 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. Mutat Res. doi:10.1016/j.mad.2011.04.012. Garcia O, Romero I, Gonzlez JE, Mandina T. Measurements of DNA damage on silver stained comets using free Internet software. (, Betti,C., Davini,T., Giannessi,L., Loprieno,N. 2). 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. Careers. Chou PH, Chen CH, Chen KY, Ko FC, Tsai TY, Yeh YP. 2023 Apr 1. doi: 10.1007/s11010-023-04720-4. Currently, the Comet Assay has however the potential to be used as a tool in genotoxicity testing and regulatory submissions for new chemicals and mixtures (Tice et al. For each treatment, the computed quartiles were compared by a 2-way ANOVA, considering the slide as the elementary unit of repetition and two random factors, electrophoresis and treatment. The Comet Assay was first introduced by Ostling and Johanson in 1984. 2014 Oct;66(5):815-22. doi: 10.1007/s10616-013-9630-y. CAS The agarose is considered to be osmotic-neutral, therefore solutions can penetrate the gel and affect the cells without cells shifting position. Assays can be conducted under neutral or alkaline (> pH 13) conditions. For all quartiles and comet parameters, the post-hoc pairwise comparisons indicate that there is no difference between the treatments control 1, control 2, -ala and visible light 30 min. The Olive tail moment and tail DNA for all the comets obtained in the different treatments and electrophoreses are presented in Figure 1. Environ Mol Mutagen 2004;44:839. There is a comet in my computer! Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Sham controls were prepared in exactly the same way, but without light exposure. (1999), the unit of comparison should be the treatment and not the comet; each series of measures has then been reduced to a statistic representative of the comet distribution.