The increasing use of single cell gel electrophoresis (the comet assay)

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA A-841720 IC50 oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their personal comet assay protocols. A-841720 IC50 Nine of 10 laboratories reported the same rating of the level of damage in the coded samples. The variance in assessment of oxidatively damaged DNA was mainly due to variations in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variance between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variance in online FPG-sensitive sites improved from 49 to 73%, whereas the inter-laboratory variance decreased. The participating laboratories were successful in finding a doseCresponse of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories. Intro Alkaline solitary cell gel electrophoresis (the comet assay) is definitely a method used to measure solitary strand breaks (SSB) and alkali-labile sites (ALS). One reason for the increasing desire for using the method is the low quantity of cells required to A-841720 IC50 measure DNA lesions. A range of different types of DNA lesions can be measured by adding lesion-specific enzymes (1). A common changes of the assay is definitely to measure the level of 8-oxoguanine as well as other modified purines by A-841720 IC50 incorporating a digestion with the bacterial DNA restoration enzyme formamidopyrimidine DNA glycosylase (FPG) (1). In addition, the comet assay can be revised to measure DNA incision activity reflecting foundation excision restoration (2) and nucleotide excision restoration (3). Several recommendations for the comet assay have been published (4C7), but there are still substantial variations in protocols used by different study organizations. These differences impact inter-laboratory comparisons of results and there is no general agreement about the normal background level of DNA lesions measured from the comet assay. Important methods in the comet assay process that may impact the variability are: (i) cell treatment/dilution in agarose, (ii) duration of enzyme treatment, (iii) duration and pH of alkaline treatment, (iv) electrophoresis conditions and (v) slip scoring. In addition, the fact that different laboratories use different end points (i.e. %DNA in tail, tail instant, tail size and arbitrary devices as well as various descriptions of the distribution of images) Igf1 when reporting their results further complicates the assessment of data between different laboratories. M?ller (8) have previously shown that there is substantial variance when different investigators score the same slides by visual classification of comets. Forchhammer (9) recently reported that inter-individual variations in visual rating could be reduced to a large extent by using investigator- and protocol-specific calibration curves. The aim of this study was to assess variance in estimations of oxidatively damaged DNA, in terms of FPG-sensitive sites, measured with the comet assay by 10 different Western Comet assay Validation Group (ECVAG) partners using their personal protocols when analyzing coded samples. Materials and methods Study design In order to investigate the inter-laboratory variance in the analysis of oxidation damage to DNA, 10 laboratories measured the level of DNA damage in four -ray irradiated calibration samples and three coded samples of human being cells using their personal protocols. The A-841720 IC50 three coded samples contained cells with different amounts of 8-oxoguanine in their DNA. Cryopreserved calibration curve samples, coded samples and aliquots of FPG from your same batch were distributed on dry ice to the participating laboratories. Laboratories were instructed to analyse the calibration samples together with the coded samples, in order to create laboratory-specific calibration curves. Each laboratory completed a questionnaire on their comet assay protocol (Table I). Table I Comet assay conditions used by the 10 different laboratories Reagents and enzymes TrypsinCethylenediaminetetraacetic acid (EDTA), Dulbecco’s Minimal Essential Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, foetal bovine serum (FBS), penicillinCstreptomycin and sodium pyruvate were purchased from Invitrogen Corporation, Karlsruhe, Germany. FPG was supplied by one of the.