Introduction to the comet assay

The comet assay, or Single-Cell Gel Electrophoresis (SCGE) Technique, was developed in the late 1980's by Swedish researchers Ostling & Johanson as a fast, effective way of measuring DNA damage in individual cells.

The Comet assay is used for a wide range of applications, including DNA damage and repair studies, genetic toxicology testing, biomonitoring and nutritional research. Perceptive Instruments are leading experts in image analysis for the comet assay. We regularly attend comet assay workshops and contribute to the debate towards regulatory acceptance of the assay.

Starting the comet assay

If you are looking to start up the comet assay within your laboratory, then why not view our brief guide on equipment you need to perform the comet assay. This page gives an introduction based on using pre-prepared commercial comet assay kits. If you intend to work with glass slides and prepare your own agarose, as many prefer to do, then our microscopy tips page will be useful as well.

Andrew Collins and his team at the University of Olso have produced a comet assay training DVD as part of the European Commission NewGeneris project FOOD-CT-2005-016320. NewGeneris is an Integrated Project conducted within the European Union's 6th Framework Programme, priority area Food Quality and Safety. Its objective is to investigate the role of prenatal and early-life exposure to genotoxic chemicals present in food and the environment in the development of childhood cancer and immune disorders.

For more information on the comet assay training DVD and on how to obtain it, please visit the News and events section of the NewGeneris website.

• Major benefits of the comet assay
• Really useful comet assay papers
• NewGeneris website

Microscopy hints and tips

If you're experiencing fading with your cells, having trouble getting your agarose to bond with your slides or just wondering which stains are best, then visit our hints page dedicated to microscopy tips for the comet assay.

Case studies

We also have a number of interesting case studies that demonstrate some of the real-world applications in which Perceptive Instruments comet assay systems are being used.

Measurement parameters and statistical analysis

The International Workshop on Genotoxicity Test Procedures recommend the use of image analysis for comet single-cell gell electrophoresis assays, and give % tail DNA, tail length and tail moment as the recommended measurement parameters. The following excerpt is from the paper that resulted from the meeting held in conjunction with the Fourth International Comet Assay Workshop:

These same recommendations were upheld at the International Workshop on Genotoxicity Test Procedures at San Francisco in 2005, with a recommendation that per cent DNA in tail be the most suitable measurement parameter:

As the comet assay is not yet a regulatory assay, there are no absolute guidelines on the reporting of results obtained. However, a number of useful papers on the subject can be found. We particularly recommend reading the following paper, the guidelines of which were used when designing the Spreadsheet Generator macro that is included with Comet Assay IV:

A more recent paper on statistics for the comet assay was published in Mutagenesis vol.23:

Recent papers: 2010

Comet Assay scoring software is used extensively in laboratories throughout the world and by leading academics in the field. We are currently compiling a list of published articles in which our Comet Assay II, III and IV systems have been utilised.

The list below shows a selection of papers from 2010. For further lists, please see our list of papers from 2009, 2008, 2007, 2006, and 2005.

A multibiomarker approach in Coris julis living in a natural environment
(2010) S. Fasulo et al, Ecototoxicol. and Environ. Safety

An ECVAG dagger trial on assessment of oxidative damage to DNA measured by the comet assay
(2010) C Johansson et al, Mutagenesis 25(2):125-132

Assessing the DNA methylation status of single cells with the comet assay
(2010) J Wentzel et al, Analytical Biochem. 400(2):190-194

Assessment of Cyto/Genotoxicity of Irinotecan in V79 Cells Using the Comet, Micronucleus, and Chromosome Aberration Assay
(2010) V Kasuba et al, Arch Ind Hyg Toxicol. 61(1): 1-9

Assessment of DNA Damage by the Comet Assay: Comparative Evaluation of Methods for Preparation of Single-Cell Suspensions from Multiple Tissues of Rats
(2010) M Manjanatha et al, Abstracts from the 8th International Comet Assay Workshop held at the University of Perugia, Perugia, Italy; 27-30 August 2009 25(1): 12

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells
(2010) V. Valdiglesiasa et al, Mut Res. 689(1):74-79

Berberine and a Berberis lycium extract inactivate Cdc25A and induce α-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis
(2010) M. Khan et al., Mutat Res. 683(1-2):123-130

Bolus ingestion of white and green tea increases the concentration of several flavan-3-ols in plasma, but does not affect markers of oxidative stress in healthy non-smokers.
(2010) N. Muller et al, Mol. Nutr. Food Res. [e-pub]

Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and non-methylating cells
(2010) E Dopp et al, Environm. Res. (in press)

Comparative Growth Performance of Mixed-Sex and Monosex Nile Tilapia Population in Freshwater Cage Culture System under Indian Perspective
(2010) SB Chakraborty & S Banerjee, Int J Biol. 2(1):44-50

Correlation between folate and vitamin B12 and markers of DNA stability in healthy men: preliminary results.
(2010) M Milić et al, Acta Biochim [e-pub]

Cyto- and genotoxicological assessment and functional characterization of N-vinyl-2-pyrrolidone–acrylic acid-based copolymeric hydrogels with potential for future use in wound healing applications
(2010) D Kirf et al, Biomed Mater. 5(3)

Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells
(2010) J Dumont et al, Tox App Pharma. (e-pub)

Diminished carcinogen detoxification is a novel mechanism for hypoxia inducible factor 1 (HIF-1) mediated genetic instability
(2010) M A Shults et al, J Bio Chem. (e-pub)

DNA Damage in Workers Occupationally Exposed to Lead, Using Comet Assay
(2010) P.Manikantan et al, Int J Biology 2(1):103-110

DNA strand breakage and lipid peroxidation after exposure to welding fumes in vivo
(2010) C.H. Chuang et al., Mutagenesis 25(1):71-76

Estimation of DNA Integrity in Blood Cells of Eastern Mosquitofish (Gambusia holbrooki) Inhabiting an Aluminium-Polluted Water Environment: an Alkaline Comet Assay Study
(2010) I Ternjej et al, Archives of Environmental Contamination and Toxicology (e-pub)

Exposure of human nasal epithelial cells to formaldehyde does not lead to DNA damage in lymphocytes after co-cultivation
(2010) S Neuss et al, Mutagenesis (e-pub)

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage
(2010) M Gavella et al, Int. J. Androl. 33(3):536-544

Genistein and β-carotene enhance the growth-inhibitory effect of trichostatin A in A549 cells
(2010) R Shiau et al, Euro J Nut. 49(1):19-25

Genomic instability in a healthy elderly population: a pilot study of possible cytogenetic markers related to ageing
(2010) M. Mladinic et al, Mutagenesis [e-pub]

Genotoxic effects of neutrophils and hypochlorous acid
(2010) N Gungor et al, Mutagenesis 25(2):149-154

Genotoxicity assessment of two vineyard pesticides in zebrafish
(2010) S. Bony et al, Int. J. Env. Analyt. Chem 90(3):421-428

Genotoxicity of anti-tumor necrosis factor therapy in patients with juvenile idiopathic arthritis.
(2010) E. Demirkaya et al, Arthritis Care Res. 62(1):73-7

In vitro effect of the antimalarial drug proguanil hydrochloride on viability and DNA damage in human peripheral blood lymphocytes
(2010) G Gajski et al, Enviro. Tox. & Pharmacol. (in press)

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay
(2010) V. Sipinen et al, Mutagenesis [e-pub]

In vitro investigations of glycidamide-induced DNA lesions in mouse male germ cells and in mouse and human lymphocytes
(2010) S H Hansen et al, Mut Res Gen Tox. 696(1):55-61

In vivo bioassay to detect irinotecan-stabilized DNA/topoisomerase I complexes in rats
(2010) S W Barth et al, Biotech J. 5(3):321-327

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules
(2010) C Hoelzl et al, Mol. Nut. & Food Res. (e-pub)

Marginal zinc deficiency increases oxidative DNA damage in the prostate after chronic exercise
(2010) Y. Song et al., Free Rad Biol Med. 48(1):82-88

Mutagenic response of 2.45GHz radiation exposure on rat brain
(2010) K K Kesari et al, Int J Rad Biol. 86(4):334-343

Nuclear phosphoinositide 3-kinase β controls double-strand break DNA repair
(2010) A Kumar et al, PNAS 107(16):7491-7496

Ofloxacin induces oxidative damage to joint chondrocytes of juvenile rabbits: Excessive production of reactive oxygen species, lipid peroxidation and DNA damage
(2010) Q. Li et al., Euro J Pharmacol. 626(2-3):146-153

Oxidative stress and DNA damage responses in rat and mouse lung to inhaled carbon nanoparticles
(2010) A. Wessels et al, Nanotox [e-pub]

Partial hydrolysis enhances the inhibitory effects of konjac glucomannan from Amorphophallus konjac C. Koch on DNA damage induced by fecal water in Caco-2 cells
(2010) Shu-Lan Yeh et al, Food Chem. 119(2):614-618

PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage
(2010) E Bobrovnikova-Marjon et al, Oncogene (e-pub)

Polyphenolic Apple Extracts: Effects of Raw Material and Production Method on Antioxidant Effectiveness and Reduction of DNA Damage in Caco-2 Cells
(2010) P. Bellion et al, J. Agricult. Food Chem 58(11):6636-6642

Polyphenolic Compounds from Salvia Species Protect Cellular DNA from Oxidation and Stimulate DNA Repair in Cultured Human Cells
(2010) A. Ramos et al, J. Agricultural Food Chem. 58(12):7465-7471

Prevention of oxidative DNA damage in inner organs and lymphocytes of rats by green tea extract
(2010) N. Kager et al, Euro. J. Nutrition 49(4):1436-6207

Protective effect of selenium supplementation on the genotoxicity of di (2-ethylhexyl) phthalate and mono (2-ethylhexyl) phthalate treatment in LNCaP cells
(2010) P Erkekoglua et al, Free Rad Bio Med. (e-pub)

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay
(2010) S Lee et al, Exp. Para. (e-pub)

Relation between DNA repair, apoptosis and chromosomal aberrations in presence of pifithrin-α, an inhibitor of p53
(2010) R Meschini et al, Mut Res. (e-pub)

Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes
(2010) P Mosesso et al, Mut Res. (e-pub)

Sensitization to Radiation and Alkylating Agents by Inhibitors of Poly(ADP-ribose) Polymerase Is Enhanced in Cells Deficient in DNA Double-Strand Break Repair
(2010) D Loser et al, Mol. Cancer Therap [e-pub]

Single exposure to cocaine or ecstasy induces DNA damage in brain and other organs of mice.
(2010) T. Alvarenga et al, Addict Biol. 15(1):96-9

Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes
(2010) G Rusak et al, Chemico-Biol. Interact. (in press)

STORAGE RESULTS IN LOSS OF THE ANTIGENOTOXIC PROPERTIES OF LENTINULA EDODES (SHIITAKE MUSHROOM) AND DEVELOPMENT OF IN VIVO GENOTOXICITY
(2010) P Alves de Lima et al, J. Food Biochem. 34(1):206-228

Subcellular Distribution of a Fluorescence-Labeled Combi-Molecule Designed to Block Epidermal Growth Factor Receptor Tyrosine Kinase and Damage DNA with a Green Fluorescent Species
(2010) M I Todorova et al, Mol Cancer Ther. (e-pub)

Synthesis and Uptake of Fluorescence-Labeled Combi-molecules by P-Glycoprotein-Proficient and -Deficient Uterine Sarcoma Cells MES-SA and MES-SA/DX5
(2010) A Larroque-Lombard et al, J. Med. Chem. 53(5):2104-2113

The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays
(2010) E Mercey et al, Biomaterials 31(12):3156-3165

The human lung cell line A549 does not develop adaptive protection against the DNA-damaging action of formaldehyde.
(2010) G. Speit et al, Environ Mol Mutagen 51(2):130-7

The influence of thermal desorption on genotoxicity of multipolluted soil
(2010) M Bonnard et al, Ecotoxicol Env Safety (e-pub)

The role of oxidative DNA damage, DNA repair, GSTM1, SOD2 and OGG1 polymorphisms in individual susceptibility to Barrett’s esophagus
(2010) E Kadioglu et al, Toxicol Ind Health. (e-pub)

Thresholds for indirect DNA damage across cellular barriers for orthopaedic biomaterials.
(2010) M Parry et al, Biomaterials [e-pub]

Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation
(2010) S Shaposhnikov et al, Tox. Let. (e-pub)

Using natural dietary sources of antioxidants to protect against ultraviolet and visible radiation-induced DNA damage: An investigation of human green tea ingestion
(2010) HM de la Roche et al, J Photochem Photobiol. (e-pub)

Vitamin C prevents DNA damage induced by renovascular hypertension in multiple organs of Wistar rats
(2010) EE Nishy et al, Hum Exp Toxicol. (e-pub)

Xanthohumol, a prenylated flavonoid contained in beer, prevents the induction of preneoplastic lesions and DNA damage in liver and colon induced by the heterocyclic aromatic amine amino-3-methyl-imidazo[4,5-f]quinoline (IQ)
(2010) F Ferk et al, Mut. Res. (in press)

XPC Silencing Sensitizes Glioma Cells to Arsenic Trioxide via Increased Oxidative Damage
(2010) Shin-Yi Liu et al, Toxicol Sci. (e-pub)