Based on our experience and that of others we recommend the use of the following for reliable and reproducible comet assay results. Comet assay software for measuring dna damage and repair. A novel dna damage response mediated by dna mismatch repair in caenorhabditis elegans. Dna damage response and repair, dna methylation, and cell. The comet assay is an efficient method to detect dna damage including single and doublestranded dna breaks. The comet assay is a wellpublished method for measuring cellular dna damage. Quantit assay kits for microplatebased assays of dna, rna. In vivo mammalian alkaline comet assay introduction 1. The assay depends on the relaxation of supercoiled dna in agaroseembedded nucleoids the residual bodies remaining after lysis of cells with detergent and high salt, which allows the dna to be drawn out towards the anode under electrophoresis. Dna damage and repair enzymes can be used in vitro to detect specific forms of dna damage. Dna repair of hydrogen peroxideinduced damage in human lymphocytes in the presence of four antimutagens. It is widely used to estimate dna damage caused by chemicals or radiation, for example, to test individual sensitivities to ionizing radiation in cancer patients.
Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive. Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique used to measure dna damage. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of dna. The essential comet assay a comprehensive guide to measuring dna damage and repair. Single cell gel electrophoresis assay comet assay is widely used for evaluating nanoparticleinduced dna damage in cells and is the most used assay for genotoxicity testing of nanomaterial. Mitochondrial and nuclear dna damage induced by curcumin in. Comet assay and foci dna damage quantification metasystems.
Advantages and limitations of the comet assay, and comparison with other assays for dna damage. This dna damage assay kit allows for duplicate analysis. The qpcr assay for analysis of mitochondrial dna damage. Detection of dna damage using comet assay image analysis for humans is noncoding, meaning that these sections donot serve a function of encoding proteins. Finally, it can be used for measurement of dna repair in vivo when employed appropriately. Detection of deoxyribonucleic acid dna damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental human biomonitoring, diagnosis of genetic disorders etc.
The increased free radicals can cause cellular damage. Adaptation to alkylation damage in dna measured by the comet assay karel j. Measuring oxidative damage to dna and its repair with the. Comet assay results, measurements and data processing. Oxiselect 96well comet assay kit cell biolabs, inc. Analysis of nanoparticleinduced dna damage by the comet.
Here, we describe the standard alkaline version of the comet assay, both in vitro and in vivo, as well as the lesionspecific enzymemodified comet assay. Dna damage including oxidative dna bases modification is one of the intrinsic signals initiating apoptosis. A study with alkaline single cell gel electrophoresis comet assay cell mol biol lett. By combining specific antibodybased detection of dna damage with a cytotoxicity indicator, both parameters can be measured simultaneously in the same cell. H2ax double strand dna damage response assay background information histone h2a variant h2a. The oxiselect comet assay is a fast and sensitive kit for the measurement of cellular dna damage. Detection of dna damage using comet assay image analysis. Increased oxidative stress in accumulated fat is an important pathogenic mechanism for obesityassociated metabolic syndrome. Each kit is supplied with appropriate primer and dna standards depending on whether you are analyzing mtdna from human, mouse or rat. We describe alkaline and neutral comet assays to measure dna damage in cancer cells to. A modification of the original comet assay was developed for the simultaneous evaluation of dna ssb and dsb in human spermatozoa.
It also permits measurement of relative mitochondrial genome copy number. In this assay, adherent cells are cultured in conventional 96 well microplates. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. Inhibition of h2o2induced dna damage in single cell gel. Dna damage can be measured as an indicator of genotoxicity using an antibody against phosphorylated h2ax.
Assaying dna damage in hippocampal neurons using the comet. In addition to measuring dna damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. The quantitative polymerase chain reaction qpcr assay allows measurement of dna damage in the mitochondrial and nuclear genomes without isolation of mitochondria. The single cell gel electrophoresis assay scge, or comet assay are sensitive method for the evaluation of dna damage. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and doublestrand dna breaks in vitro. We primarily use the comet moment, defined by kent et al. The aim of the present study was to detect the type of dna damage and cell death caused by ionizing radiation as well as the sensitivity of the standard and modified comet assay. Ionizing radiation ir can result in serious genomic instability and genotoxicity by causing dna damage. Diagnosis of metals induced dna damage in fish using comet. The dna in a human cell undergoes several thousand to a million damaging events per day, generated by both external exogenous and internal metabolic endogenous processes.
Single cell gel electrophoresis assaycometassay a simple and effective method for evaluating dna damage in single cells. The epiquik in situ dna damage assay kit is a convenient set of tools that allows the experimenter to detect dna damage or apoptosis by measuring phosphorylation of h2ax ser9 in situ. The comet assay is a quantitative method for measuring dna damage primarily strand breaks. Dna damage was assayed by using the comet assay and dna gel electrophoresis and the results indicated that ncih460 cells treated with 0, 50, 250 and 500 nm deguelin led to a longer dna migration smear based on the single cell electrophoresis and dna fragmentation occurred based on the examination of dna gel electrophoresis. The assay depends on the relaxation of supercoiled dna in agaroseembedded nucleoids the residual bodies remaining after lysis of cells with detergent and high salt, which allows the dna. Dna damage is a precursor of cancer, but the link is a weak one, as almost all damage.
The alkaline comet assay was used to determine ndna fragmentation. Development of a qpcr method to measure mitochondrial and. Keywords increase sensitivity with confocal acquisition, highthroughput imaging assay for detecting dna damage, detecting dna damage. Dna repair in plants studied by comet assay karel j. Quantit assay kits for microplatebased assays of dna, rna, and protein selective quantitationmore accurate than uv absorbance readings or bradford assays high sensitivityuse as little as 1 l of your sample simple assay designmixandread format for quantitation in microplates, with or without robotics.
Induction of dna damage by deguelin is mediated through. Conclusion this is the first attempt to use alkaline comet assay to evaluate dna damage in bangladeshi bladder cancer patients. Mutation research 493 2001 8793 dna damage and repair in arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins merten menkea, ipeng chena. Cells were detached from culture plates by incubation with 0. Once you have obtained your results, you can easily process them using the spreadsheet generator, which is included free with every comet assay iv system. All the comet assay results files produced by the comet assay iv system can be used with the excelbased spreadsheet generator, which is supplied free with every comet assay iv system.
The investigation of dna damage and repair at the single cell level can be performed, in a very. Comet assay is reliable, fast, capable of detecting low level of dna damage, requires short time and is a responsive technique for single or double strand breakage in dna, cell death, inter strand. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the dna and convert lesions to dna breaks, increasing the amount of dna in the comet tail. Sensitivity with confocal acquisition, imaging assay for. The singlecell gel electrophoresis assay comet assay is a method highly sensitive to dna damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. It also has applications in assessing the antioxidant status of cells. The comet assay single cell microgel electrophoresis was originally developed for the detection of mutageninduced dna damage 14 such as by electric field dna fragments or loops are extruded from agaroseembedded nuclei of single cells. After treating dna containing ap sites with arp reagents, ap sites are tagged with biotin residues, which can be quantified using avidinbiotin assay. As the conceivable cause of dna damage, the level of ros and lipid peroxidation were analyzed. I have already evaluated the dna damage by comet assay. The comet assay is an extremely sensitive dna damage assay.
Dna damage quantification colorimetric kit k253 biovision. H2ax assay is based on the total phosphorylation of h2ax histone in response to dna damage by induction of doublestrand breaks dsbs. Nucleic acid, protein quantitation, picogreen, hoechst 33258 dye, quantit dsdna assay, quantit ssdna. Under an electrophoretic field, damaged cellular dna containing fragments and strand breaks is separated from intact dna. Dna repair measured by the comet assay, dna repair, inna kruman, intechopen, doi. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of dna damage in all cell types, and combines the simplicity of biochemical techniques for detecting dna. Assay principle the cell biolabs oxiselect comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage. O 2uv dna damage normal cell dna repair damaged ageing cell ber ner. While adobe acrobat can be a great help to work around limitations of pdf file forensics and analysis, a lot of jobs can be simplified using external pdf forensics tool. Limitations of the comet assay, and misunderstandings it is wise to point out the limitations of the comet assay. Comprehensive measurements and results easily graph and report your data. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Few genotoxic agents break dna directly, but many lesions can be converted to breaks, and dna repair pathways involve breaks as intermediates. Usage of the standard and modified comet assay in assessment.
Finally, dose responses for dna damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and. X, a component of the nucleosome core structure has a special role in dna repair. The comet assay allows testing of a broad spectrum of dna damage with high sensitivity, in vitro as well as in vivo 3, 4. Dna damage is quantified by the proportion of dna which migrates out of the nuclei.
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. Measuring of dna damage by quantitative pcr ayse gul mutlu mehmet akif ersoy university, arts and sciences faculty, department of biology, burdur turkey 1. After analysis of a sufficient number of nuclei, the data are analyzed with appropriate methods to judge the assay. Dna damage and cell death assessment in patients with. The comet assay in toxicology issues in toxicology. These dna damage assay kits are for the determination of damaged 8. Automated analysis of dna damage in the highthroughput. Electrophoresis at high ph results in structures resembling comets, observed by. The in vivo alkaline comet single cell gel electrophoresis assay hereafter called simply the comet assay is used for the detection of dna strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic materials. Ber mechanism is used when dna is affected by reactive oxygen species, alkylating agents by oxidation or single strand break. After your experimental treatment, cells are fixed and permeabilized. In repair experiments residual dna damage could be. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the dna and convert lesions to dna breaks, increasing the amount of dna.
At present, data about dna damage in critical illness are lacking. The kit is readytouse and provides all the essential components needed for specifically measuring dna damage. The two strands of dna run in opposite directions to each other and are hence antiparallel. The results of this study may emphasize to use aca for routine dna damage detection, which could be used as a prognostic biomarker of bladder cancer. Inhibition of h2o2induced dna damage in single cell gel electrophoresis assay comet assay by castasterone isolated from leaves of centella asiatica nishi sondhi1, renu bhardwaj1, satwinderjeet. Each kit provides sufficient reagents to perform up to 75 assays. In vivo mammalian alkaline comet assay introduction. As the cells are subjected to the electrical field, the dna migrates at different rates due to differences in size which is subsequently analyzed using the comet assay software. Comet assay, dna damage and mutagenesis, nutrition and cancer, dna repair in.
There are several endpoints for the comet assay but the dna content in the tail % tail dna or % tail intensity is recommended to assess dna damage 12. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. Application methods p a g e 1 080409 nucleic acid and protein quantitation methods prepared by. Thus, this lqpcr dna damage assay can be used to screen drug. Dna kits rna kits protein kits dna damage kits apoptosiskits elisa kits cell migrationinvasion kits angiogenesis kits oxidative stress kits enzyme activity assays other kits. In addition, sensitivity of both techniques toward different doses of gamma radiation was measured. During the past two decades the cometbased in vitro dna repair assay. X by pi3klike kinases, including atm, atr and dnapk, is an early cellular response. The comet assay for dna damage and repair springerlink. Evaluation of storage conditions for assessing dna damage using the comet assay dante villavicencio submitted to the facultyof the universitygraduate schoo l in partial fulfillment of the requirements for the degree master of science in the department of pharmacologyand toxicology, indiana university october 2006. Dna damage in brain cells of rats exposed to high f and low i 211 fluoride 2005. Measurement of dna damage in bladder cancer patients by. Damage to cellular dna is involved in mutagenesis and the development of cancer. The comet assay single cell gel electrophoresis is the most common method for measuring dna damage in eukaryotic cells or disaggregated tissues.
Page 1 of 4 colorimetric assay kit for dna damage quantification for research use only introduction during the course of dna excision and repair of oxidized, deaminated or alkylated. A novel dna damage response mediated by dna mismatch. Article a twotailed comet assay for assessing dna damage. The comet assay for dna damage and repair applications in.
It can usually be performed on materials found in most laboratories. Since the initial development of the comet assay, efforts have been made to improve assay sensitivity and reliability, extend applications to the analysis of various types of dna damage in various cell types, increase samplehandling capacity, and standardize the protocols and analysis. The dna damage quantification kit utilizes the arp aldehyde reactive probe reagent that reacts specifically with an aldehyde group which is the open ring form of the ap sites. Since the initial development of the comet assay, efforts have been made to improve assay sensitivity and reliability, extend applications to the analysis of various types of dna damage. The comet assay, or single cell gel electrophoresis assay scge, is a common technique for measurement of dna damage in individual cells. Bioseparation bioseparation reagents solvents bioseparation media electrophoresis instruments stains and markers precast gels agarose acrylamide electrophoresis buffers. The dna damage quantification kit utilizes the arp aldehydereactive probe reagent that reacts specifically with an aldehyde group which is the openring form of the ap sites. Pdf file forensic tool find evidences related to pdf. In this case, irradiation of the neuronal cells induces dna damage. Comet assay iv combines accurate, repeatable results with powerful data processing and comprehensive presentation. Dna damage and obesity in diabetic patients gursatej gandhi1 and amanjit kaur saini1 research paper objective. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, noninvasive, inexpensive, direct and sensitive measure of dna damage and repair.
Comet assay iv tm is the latest in a highly successful series of comet scoring systems originally developed by perceptive instruments, now a part of instem. The hcs dna damage kit uses a secondary antibody conjugate to detect phosphorylated h2ax, imageit dead green dye to detect. A comet assay for dna damage and repair after exposure to. All forms of dna damage as well as dna repair can be visualized at the single cell level using this powerful technique. We describe alkaline and neutral comet assays to measure dna damage in cancer cells to evaluate the therapeutic effect of chemotherapy. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. Dna damage is a precursor of cancer, but the link is a weak one, as almost all damage is repaired. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance getting started with comet assay. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing.
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. We also present techniques used to extend the assays dynamic range by removing interfering background fluorescence and to define a region of interest. As shown above as a bar graph, cox2dependent dna damage dramatically increased with bpd treatment. Dna damage and repair in arabidopsis thaliana as measured. Assessing dna dam age in marine species through singlecell alkaline gel electrophoresis comet assay. A high sensitivity, high throughput, automated singlecell. The 2tcomet assay is a fast, sensitive, and reliable procedure for the quantification and characterization of dna damage. Npstio2 and lincomycin coexposure induces dna damage in. Considering that, two forms of the comet assay, standard alkaline and fpgmodified were used. The comet single cell gel electrophoresis assay is a method for obtaining the amount of dna fragmentation of single cells.
Evaluation of dna damage using singlecell gel electrophoresis comet assay the comet assay, also known as singlecell gel electrophoresis scge, is a simple, sensitive and reliably efficient method for studying dna damage induced by. A high sensitivity, high throughput, automated singlecell gel electrophoresis comet dna damage assay. The comet assay is a dna damage quantification method that is efficient and easy to perform, and has low timebudget demands and high reproducibility. The dna damage in the liver and kidney cells of mice was evaluated by the fpgmodified comet assay which revealed a significant p dna lesions in liver. After treating dna containing ap sites with arp reagents, apsites are tagged with biotin residues, which can be quantified using avidinbiotin assayfollowed by a. Our data suggested that low doses of curcumin did not damage dna, and at the low doses, curcumin could play an antioxidant role in carcinogenesis. Genotoxic effect induced by hydrogen peroxide in human. Evaluating in vitro dna damage using comet assay protocol.
Jun 27, 2006 we present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. The spreadsheet generator is provided to help generate graphs and tables in excel from the raw data produced by the comet assay. The comet assay is a suitable technique to measure various types of dna damage and their repair. It has proved its usefulness and versatility in human biomonitoring, ecogenotoxicology, genotoxicity testing, and basic research into the mechanisms of dna damage and repair. Comet assay single cell gel electrophoresis sigmaaldrich. By using the comet assay to quantify dna damage, we demonstrate that combining olaparib with temozolomide profoundly enhances the dna damage in glioma cells, which suggests olaparibtemozolomide combination therapy is an effective strategy to treat glioma, as compared with temozolomide alone11. Angelis, jaroslav kozak, radka vagnerova and marcela hola institute of experimental botany as cr, czech republic institute of organic chemistry and biochemistry as cr, czech republic 2015 icaw antverpen.
As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Comet assay does not require any special apparatus or chemicals. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet cinduced dna damage was carried out. Assessing dna damage in marine species through singlecell. Each kit contains a mitochondrial dna standard, dna. For determining dna damage, the comet assay is a wellestablished genotoxicity test that offers the possibility of measuring in a highthroughput mode. In addition to the cell system, the lqpcr dna damage assay can be used with tissue or blood specimens, fresh or frozen. May 18, 20 the comet assay single cell gel electrophoresis is the most common method for measuring dna damage in eukaryotic cells or disaggregated tissues. This is not the case for standard cytogenetic methods that, due to heterogeneity in genome damage caused by ionizing radiation, provide only average dna damage information. Assaying dna damage in hippocampal neurons using the comet assay. Cometassay in combination with pnafish detects mutagen. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit.
368 685 126 1268 1557 1091 1363 81 822 731 585 152 928 48 875 1408 1333 1245 1438 432 1130 1174 653 73 531 1427 1553 242 1563 1334 433 1520 884 256 1144 845 150 337 1444 587 1398 1215