Development of a digital analysis method to comet assay (silver staining)

Abstract

The comet assay is a genotoxicity test which use is still evolving, in spite of be performed for more than 20 years. It is a fast method, low cost, of relative accomplishment easiness, safe and it can be applied in several tissues. It is based in the migration estimate of submitted cellular DNA by an electrophoresis current. The staining process can be accomplished alternatively by fluorescence techniques or silver staining. The two colorations do not differ in relation to the results of the obtained images, therefore, being equivalent. The automated analysis method of the comet assay silver staining still was not standardized and it needs a validation of the process. The analysis can be performed in several ways, one of them is the accomplishment of measures of morphologic parameters that it can be carried out in a manual way or for systems of image analysis automated. These systems allow obtaining less subjective results, with a larger reproducibility and precision. For the accomplishment of these measures by the automated method, digital pictures of the comets are needed. The capture of these images is a very important stage and it should be executed in a standardized way. For the analysis of digital image it is necessary series of processing to make possible the segmentation of the image in relation to the background and the quantification of the DNA damage. The authors propose the development and validation of a digital analysis technique for the comet assay silver stained. (AU)