The adoption of pcrbased techniques to quantify dna damage has enabled sequence and organellespeci. The comet assay is a dna damage quantification method that is efficient and easy to perform, and has low timebudget demands and high reproducibility. By combining specific antibodybased detection of dna damage with a cytotoxicity indicator, both parameters can be measured simultaneously in the same cell. To quantify the induced dna damage, 100 cells per probe were examined for the olive tail moment otm reflecting the percentage of dna in the tail of the comet multiplied by the median migration distance 19, the percentage of dna in the tail %dt, tail moment tm and the tail length tl 20. Damage to dna, including oxidative dna damage, has been implicated in the development of a variety of cancers including colon, breast and prostate. The comet assay for dna damage and repair applications in. The comet assay is a versatile and sensitive method for measuring single and doublestrand breaks in dna. Dna damage response deficiency assay predicts response to treatment in ovarian cancer laura a. Read online comet assay of dna damage in brain cells of adult rats. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparibtemozolomide combination therapy to u251 glioma cells.
Dna damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. Investigating oxidative dna damage and its repair using. The main goal of this study was to investigate antigenotoxic potential of a standardized dry olive leaf extract on dna damage induced by 17. All books are in clear copy here, and all files are secure so dont worry about it. Dna damage quantification kit contains all the necessary solutions, enabling the determination of 1 to 40 ap sites per 1x105 bp. Dna damage kits 0 amsbio specialise in dna damage assay kits, enzymes and antibodies, to measure dna damage universally in cells and tissues and at a molecular level. No extract plus extract rate of accumulation of breaks is a measure of ogg repair activity.
Detection of dna damage using comet assay image analysis. In the simplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution, and then exposed to an electric. Description of the kit dna damage quantification kit ap site counting is for the determination of abasic sites in sample dna, which correspond to colorimetric 96well microplate assay. Detection of dna doublestrand breaks through the cell.
Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. The aim of this study is to determine whether toxoplasmosis caused by rh virulent strain would cause dna damage with assessment of its extent in. Reactive species such as free radicals are constantly produced in vivo and dna is the very important target of oxidative stress. Typically, neutral comet is used to detect dna doublestranded breaks while alkali comet detects a wider breadth of dna lesions including dna singlestranded breaks and abasic sites. A modified version of the challenge assay, the cometfish assay a combination of the comet assay with fluorescent in situ hybridization fish, using labeled probes to particular dna sequences has been used to study dna repair of single genes or dna sequences shaposhnikov et al. Epiquik 8ohdg dna damage quantification direct kit. Health benefits of dark coffee protecting blood cells from dna damage created. Single cell electrophoresis assay also known as comet assay is a rapid, simple, visual and sensitive technique for measuring and analyzing dna breakage in mammalian cells2123.
Xaxis is the number of ap sites and yaxis is the o. Dna damage quantification assay kit colorimetric nbp2. The two strands of dna run in opposite directions to each other and are hence antiparallel. The comet assay is a powerful tool for the assessment of dna integrity with applications as diverse as cancer research, safety testing of pharmaceuticals and chemicals, environmental and. Comet assay interest group is a free forum for exchange of information on comet assay worldwidejoining this group you will have free access to the latest developments in this field as well as access to archived discussions at nih listserv. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. The comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th anniversary of. 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. Thisisimportant because dna damage can lead to cancer and other diseases.
Comet assay single cell gel electrophoresis sigmaaldrich. Automated analysis of dna damage in the highthroughput. Mutations to checkpoint kinases can ultimately lead to decreased dna repair and increased susceptibility to cancer. To determine whether the power of the comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity. Oxidative stress has been shown to be the greatest factor in dna damage, and increased levels have been closely correlated to harmful environmental factors such as ionizing radiation. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells.
We demonstrated that in a rat thyroid cell line pccl3, nonlethal concentrations of h 2o. 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. 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. Although the comet assay, a procedure for quantitating dna damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. M p comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. As shown above as a bar graph, cox2dependent dna damage dramatically increased with bpd treatment. Dna damage can be measured as an indicator of genotoxicity using an antibody against phosphorylated h2ax. The dna damage quantification kit utilizes the arp aldehyde reactive probe reagent that reacts specifically with an aldehyde group which is the openring form of the ap sites. Download dnadamage poster the human genome is exposed to potentially deleterious genotoxic events during every cell division cycle. Dna damage response deficiency assay predicts response. Oxidative dna damage is considered as a predictive biomarker to monitor the risk of development of many diseases. The comet assay singlecell gel electrophoresis is a simple and sensitive method for studying dna damage and repair. You can also send your messagequeries to the entire group.
The 2tcomet assay is a fast, sensitive, and reliable procedure for the quantification and characterization of dna damage in spermatozoa. Our results indicated that both hormones showed a genotoxic effect at a concentration of 100. It is a valuable tool in population monitoring, for example in assessing the role of oxidative stress in human disease, and in monitoring the effects of dietary antioxidants. Regardless of the nature or location of the dsbs, chinese hamster v79 cells with sphase dna content showed about 23 times less damage by all agents than cells with g1 or g2mphase dna content. Evaluating in vitro dna damage using comet assay protocol. Trevigens comet analysis software can rapidly evaluate large.
Scores of dna damage, in terms of strand breaks, were obtained by comet assay alkaline condition for singlestrand breaks ssbs and neutral condition for dsbs. Article a twotailed comet assay for assessing dna damage. Thus, this lqpcr dna damage assay can be used to screen drug. The hcs dna damage kit uses a secondary antibody conjugate to detect phosphorylated h2ax. After treating dna containing ap sites with arp reagents, ap sites are tagged with biotin residues, which can be quantified using avidinbiotin assayfollowed by a. In this microgel electrophoresis technique, a small number of. Recently oxidative rna damage has been described in conjunction with a wide variety of neurological diseases. The comet assay, or single cell gel electrophoresis scge, is well established as a direct measure of dna damage in genotoxicity studies. Dry olive leaf extract attenuates dna damage induced by. The epiquik 8ohdg dna damage quantification direct kit colorimetric is a complete set of optimized buffers and reagents to colorimetrically detect and quantify oxidative dna damage 8ohdg directly using dna isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance getting started with comet assay iv is as simple as installing the software and connecting the camera. Dna damage is commonly measured at the level of individual cells using the socalled comet assay singlecell gel electrophoresis. Alkaline treatment electrophoresis dna damage to substrate cells in vitro assay for ber % dna in comet tail indicates break frequency.
As the frequency of dna breaks increases, so does the fraction of the dna extending towards the anode, forming the comet tail. The comet assay can be used to detect dna damage caused by double strand breaks, single strand breaks, alkali labile sites, oxidative base damage, and dna crosslinking with dna or protein. It is an innovative method for assessing sperm dna integrity, which has important implications for human fertility and andrological pathology. The comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th anniversary of the first description of this methodology. For the measurement of dna double strand breaks cells were lysed in a buffer containing 30mm. Genotoxicity assessment of saline extract from pilosocereus gounellei cactaceae and its chemopreventive effect against cyclophosphamideinduced dna damage. A high sensitivity, high throughput, automated singlecell.
We supply ready to use assays for parp, parg, and par, including a range of kits, cometslides, control cells and electrohoresis tanks. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. In addition to the cell system, the lqpcr dna damage assay can be used with tissue or blood specimens, fresh or frozen. Residual protein content measured after lysis of sphase cells embedded in agarose did not differ. The comet assay for dna damage and repair springerlink. It has since increased in popularity as a standard technique for evaluation of dna. The software applies state of the image processing methods to automatically locate and score comets to characterize and quantify the degree of dna damage revealed by the comet assay. A high sensitivity, high throughput, automated singlecell gel electrophoresis comet dna damage assay.
Dna damage quantification kit ap site counting technical. The mechanism of formation of comets under neutral or alkaline conditions is best understood by analogy with nucleoids, in which relaxation of dna supercoiling in a structural loop of dna by a single dna break releases that loop to extend into. The comet assay is a gel electrophoresis method used to visualize and measure dna strand breaks in individual cells, using microscopy. 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 comet assay is not the only way to measure oxidative dna damage, but it is one of the most sensitive and accurate, being relatively free of artefacts. The comet assay is a wellpublished method for measuring cellular dna damage. Original article dna damage induced by tartrazine in rat. Download comet assay of dna damage in brain cells of adult rats. 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. Hydrogen peroxide induces dna single and doublestrand.
The comet assay single cell gel electrophoresis is the most common method for measuring dna damage in eukaryotic cells or disaggregated tissues. Use of single cell gel electrophoresis comet assay. Electrophoresis at high ph results in structures resembling comets, observed by. Dna damage quantification kitap site counting dojindo. Development of a qpcr method to measure mitochondrial. This endogenous source of dna damage results from cellular metabolism or routine errors in dna replication and recombination.
Evaluation of dna damage by dna fragmentation and comet. Is the comet assay a sensitive procedure for detecting. In this regard, parp is specifically cleaved by caspases so that it is no longer active, preventing dna repair and promoting apoptotic processes. 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 comet assay allows testing of a broad spectrum of dna damage with high sensitivity, in vitro as well as in vivo 3, 4. The comet assay is widely used for specifying oxidative dna damage at a single cell level. 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. Automated comet assay imaging and dualmask analysis to. In the simplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution, and then exposed to an electric field to attract.
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