Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results

Abstract

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

Fig. 1. Scheme of the comet assay procedure.

The comet assay consists of nine steps, which are described in the text and in Table 1. Tissues or cells are isolated and processed to a single-cell suspension, either to study DNA damage (Step 1A) or to prepare substrate cells for the in vitro DNA repair assay (Step 1B). Next, single cells are embedded in agarose gels (Step 2) and lysed (Step 3) to remove membranes and other cellular material, leaving protein‐depleted nuclei with supercoiled DNA (called ‘nucleoids’). Step 4 does not apply to the standard alkaline comet assay, but comprises specific steps for the enzyme-modified comet assay (i.e., incubation of the nucleoids with lesion-specific enzyme; Step 4A) or in vitro DNA repair assay (i.e., incubation of the substrate cell nucleoids with cell or tissue extract; Step 4B). For the enzyme-modified comet assay, possible enzymes include formamidopyrimidine DNA glycosylase (Fpg), human oxoguanine DNA glycosylase (hOgg1), endonuclease III (Endo III) and T4 endonuclease V (T4 Endo V). In Step 5, samples are treated in alkaline solution to convert alkali-labile sites to strand breaks. The samples are then subjected to alkaline electrophoresis, resulting in the formation of single-cell comets (Step 6), and then rinsed in neutralizing solution (Step 7). Step 8 includes staining and visualization of the comets by fluorescence microscopy. Examples of comets include nucleoids that have been incubated with buffer in Step 4 (i.e., DNA strand breaks or background control for the enzyme-modified comet assay and in vitro DNA repair assay, respectively). Nucleoids may show clear comet formation if the DNA in the sample contains many lesions or the cell extract has high repair activity. Conversely, sparse DNA lesions and low repair activity give rise to comets that are no different from those for the background control. The comets are then scored, and finally data analysis is performed (Step 9).