Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results — TEI-tagged view

Peter MøllerpersName, Amaya AzquetapersName, Elisa Boutet-RobinetpersName, Gudrun KoppenpersName, Stefano BonassipersName, Mirta MilićpersName, Goran GajskipersName, Solange CostapersName, João Paulo TeixeirapersName, Cristiana Costa PereirapersName, Maria DusinskapersName, Roger GodschalkpersName, Gunnar BrunborgpersName, Kristine B. GutzkowpersName, Lisa GiovannellipersName, Marcus S. CookepersName, Elke RichlingpersName, Blanca LaffonpersName, Vanessa ValdiglesiaspersName, Nursen BasaranpersName, Cristian Del Bo’persName, Bojana ZegurapersName, Matjaz NovakpersName, Helga StopperpersName, Pavel VodickapersName, Sona VodenkovapersName, Vanessa Moraes de AndradepersName, Monika SramkovapersName, Alena GabelovapersName, Andrew CollinspersName, Sabine A. S. LangiepersName (2020date). “Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results.” Nature Protocolstitle·j.

Object of study —

Research question — which procedural details are essential or desirable for interpreting and reproducing comet-assay results

Contribution — the MIRCAterm standardized reporting checklist

Full text — tagged

The reference list is not part of the text we hold for this paper. Our copy was extracted from the publisher's page and the bibliography did not survive that extraction, so the article below ends before its references. Nothing has been removed by us.

Abstract. Abstract The comet assayterm 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 assayterm procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assayterm (MIRCAterm) providing recommendations for describing comet assayterm 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 MIRCAterm recommendations should ensure that comet assayterm results can be easily interpreted and independently verified by other researchers. Subject terms: Biomarkers, Risk factors Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assaysterm (MIRCAterm). Introduction The alkaline comet assayterm is a technically simple, sensitive assay to detect DNA damage (strand breaks and other lesions that are converted into strand breaks under alkaline conditions) and DNA repair activityterm·def 1 – 4 . However, international ring trials have identified substantial variations in comet assayterm procedures and primary descriptors (such as %DNA in tail) between laboratories. This variation could hamper inter-laboratory data comparison and interpretation, as well as attempts to standardize methods and promote the use of reference standards 5 – 14 . A recent review summarized the plenitude of procedure descriptions and technical recommendations for comet assaysterm that have been published in the past 20 years, and highlighted the problem of inter-laboratory variation in DNA damage levels 15 . The main issue is that the comet assayterm does not directly measure the number of specific DNA lesions, but rather measures the migration of DNA in agarose gels as a result of the relaxation produced by strand breaks under alkaline conditions. Certain steps in the assay procedure are more important determinants of DNA migration than others. However, published comet assayterm studies often inadequately describe assay conditions. Even more troubling is the lack of data on positive experimental controls and positive assay controlsterm·def, which are necessary for assessing assay performance 16 , 17 . The OECD guideline on the in vivo comet assayterm (TG489), which was developed by multiple authors at various different institutions, is the most authoritative set of recommendations for the reporting of in vivo comet assayterm procedures and results 18 . However, it does not cover in vitro experiments and biomonitoring studies, or endpoints other than DNA strand breaks, and the level of detail is limited in some aspects. For example, the guidelines recommend that “electrophoresis conditions” should be reported but without specifying that these should include the composition and temperature of the electrophoresis solution, as well as the duration of electrophoresis and strength of the field. There is therefore an urgent need for a more comprehensive set of recommendations to describe comet assayterm conditions, including explanations for why certain steps need to be reported in detail. Similar recommendations have been published for other types of assays, such as the MIAME guidelines for microarrays 19 and the MIQE guidelines for quantitative real-time polymerase chain reaction procedures 20 . These have been widely adopted by authors and journals, and appear to have improved the harmonization of reporting and the reliability of the results. The goal of the Minimum Information for Reporting Comet Assayterm (MIRCAterm) recommendations is to highlight key aspects of the comet assayterm procedure that must be described when reporting the results from cell culture studies, animal models, invertebrates, plants and human biomonitoring and clinical studies. We acknowledge that every step in the protocol is important for a well-functioning method. However, the purpose of the MIRCAterm recommendations is to ensure that specific information about the comet assayterm procedure is available to readers that will allow the results to be interpreted critically and compared with those from other studies. We have restricted the MIRCAterm recommendations to the technical performance of the assay. There are other fundamental aspects of conducting and reporting scientific studies (e.g., blinding of samples, minimizing bias and confounding factors, reporting brands and suppliers of chemicals, kits and laboratory equipment, ensuring an appropriate number of replicates and method of statistical analysis, etc.), but these are not unique to comet assaysterm and are not discussed in detail here. For the use of the comet assayterm in molecular epidemiology studies, we recommend reading the Strengthening the Reporting of Observational Studies in Epidemiology–Molecular Epidemiology (STROBE-ME) statement and following the recommendations there for reporting biomarker results 21 . Overview of MIRCAterm recommendations The MIRCAterm recommendations focus on the steps in the comet assayterm that may affect the level of DNA migration (e.g., a physical effect that causes DNA to move faster in agarose irrespective of the absolute amount of DNA damage) or increase the difference in DNA migration between unexposed and exposed specimens (i.e., detection of induced DNA damage). The variation in comet assayterm procedures has been discussed previously 15 and will not be described in detail here. Most comet assaysterm determine DNA damage as frank strand breaks (and alkali-labile sites, which are subsequently converted to strand breaks) by the standard alkaline procedure. However, DNA repair enzymes from bacteria or human cells can also be used to gain further information on specific classes of DNA lesions such as oxidation or alkylation products. The two most commonly used modified comet assayterm formats for DNA repair are (i) the ‘cellular DNA repair assayterm’, in which the accumulation and removal of DNA damage is followed over time, and (ii) the ‘comet-based in vitro DNA repair assayterm’, which measures the DNA incision activity of a cell-free protein extract on substrate DNA (in the form of nucleoids) containing DNA lesions. The ‘neutral comet assayterm’ (whereby electrophoresis is carried out in a solution of neutral pH) is rarely used 22 , 23 and is therefore not included as a separate protocol step in the MIRCAterm recommendations. Irrespective of the cell or tissue sample being investigated, the comet assayterm has up to nine steps (Fig. 1 ), as follows: (i) isolation of cells and preparation of single-cell suspensions, (ii) embedding of the cells in agarose, (iii) cell lysis, (iv) incubation of the nucleoids with lesion-specific enzyme (for the enzyme-modified comet assayterm) or with cell or tissue extract (for the in vitro DNA repair assay), (v) alkaline treatment, (vi) electrophoresis, (vii) neutralization, (viii) staining and visualization and (ix) scoring and data analysis. Table 1 outlines the MIRCAterm recommendations for these individual steps. Each recommendation is classified as either ‘desirable’ information or ‘essential’ information to be reported in articles that include comet assayterm results: ‘essential’ information is necessary for assessing the quality of the comet assayterm experiments, whereas ‘desirable’ information is only needed for repeating the experiment. Fig. 1 Scheme of the comet assayterm procedure. The comet assayterm 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 assayterm, but comprises specific steps for the enzyme-modified comet assayterm (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 assayterm, 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 assayterm 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). Table 1 Reporting checklist for comet assayterm studies Step Comet assayterm parameter Reporting requirement a Notes and rationales 1A Isolation of cells Preparation of a single-cell suspension (from solid tissue or cell culture) Desirable The homogenization procedure (whether in buffer or medium) may affect levels of DNA damage. Cell type Essential For human biomonitoring studies, it should be specified whether the samples are whole blood (i.e., with erythrocytes), isolated leukocytes or peripheral blood mononuclear cells, or from which organ or tissue the cells are derived (buccal, sperm, etc.). Method for venipuncture and isolation of cells from blood (if cells were isolated) Desirable Expected to be of little importance in most cases, but the gauge of needle and anticoagulant used might affect the level of DNA damage during cell isolation. Temperature and duration of transfer from isolation of cells to processing of cells Essential The temperature and time period between isolation of cells and direct processing in comet assayterm (or cryopreservation) may affect the level of DNA damage. Storage (in case of specimens that have been cryopreserved) Essential The freezing and thawing procedures might increase the basal level of DNA migration. For clinical intervention studies, it is essential to know whether samples taken at different times were analyzed fresh (i.e., in different experiments) or in the same comet assayterm experiment in the case of cryopreserved samples. 1B Substrate cells (for DNA repair assay only) Substrate cell type Desirable The DNA content and chromosome structure differ between different immortalized cells (cell lines) and between primary and immortalized cells. Cell density Desirable The in vitro DNA repair assay measures the rate of incisions, where the amount of enzyme is the limiting factor. Theoretically, if the DNA migration in each comet depends on number of incisions, increasing the cell density will dilute the effect by yielding fewer incisions per comet. Type of exposure used Essential Very few (if any) genotoxic agents give rise to DNA lesions that are repaired by only one DNA repair pathway; rather, most give rise to a spectrum of DNA lesions. The concentration/dose of the genotoxic agent should be reported. Levels of lesions in the substrate cells Desirable It is desirable to know the total number of lesions in the substrate cells because the repair incision activity must be measured under conditions in which the concentration of substrate (lesions) is not rate limiting (in keeping with basic enzymology). Storage (in case specimens have been cryopreserved) Essential See same item under section 1A. 1C Assay controlsterm·def Essential Assay controlsterm·def should always be included and reported in studies that do not have a positive control group. 1D Negative and positive controlsterm·def Desirable Control groups are desirable (or even essential in certain cases). For most purposes, however (and especially in human biomonitoring), assay controlsterm·def can replace negative and positive controlsterm·def (i.e., control groups). 2 Embedding the cells in the agarose Description of the type of slides Desirable Use of 2-gel versus 12-gel format, etc., might affect the level of DNA migration. Final concentration of low-melting-point-agarose containing cells Essential The final concentration (percentage after the cells have been added) is very important. As the concentration will change upon reuse of the agarose stock solution, it should be specified if it is used more than once. It is not informative enough to state the concentration of the stock solution. 3 Lysis Buffer composition Essential For buccal cells, an extra lysis step with proteinase K is needed. Lysis of sperm requires an incubation step with dithiothreitol and proteinase K to break disulfide bonds in the tightly packed DNA. Duration Desirable The duration of the lysis can vary depending on the cell type. If it is too long, this may affect certain types of DNA lesions (e.g., conversion of alkali-stabile lesions to strand breaks), and if too short, lysis might be incomplete. It is important that the same duration is used in all experiments. Temperature Desirable Expected to have little effect on DNA migration, except in certain cases where alkali-stabile lesions may be converted to strand breaks. 4A Enzyme treatment Washing step between the lysis and the enzyme treatment Desirable The enzymes may be inactivated by carryover of the lysis solution, due to high pH and detergents. The composition of the wash buffer should be specified. Source of repair enzyme Essential There are different manufactures of enzymes for the comet assayterm, which can be obtained as crude extracts or purified enzymes. Thus, the enzyme activity may vary between manufactures. Optimization of enzyme concentration and duration of incubation Desirable Authors should report or reference the results from a titration experiment using the same gel incubation unit and mode of incubation as the test samples. Duration Essential The number of repair incisions is proportional to the incubation time. However, prolonged incubation times may lead to nonspecific incisions. Incubation temperature Essential The rate of enzymic reactions depends on the temperature. Concentration of enzyme applied onto gels Essential The amount of enzyme on the gel will affect the number of repair incisions. It is important to report the results from optimization experiments (i.e., the amount of enzyme and the duration of the incubation period). Type of incubation unit Desirable Incubation in a regular incubator, slide moat or 12-gel system can all give different results. Mode of incubation Desirable The treatment is done either by dropping the enzyme solution onto the gels and covering with a coverslip or by immersing the slide in the enzyme solution. Differences in resulting enzyme activity have been noted, although this has not been assessed in a systematic manner. 4B Extract preparation/incubation (for DNA repair only) Number of cells or milligrams of tissue used to prepare the extract Desirable The number of cells or weight of tissue used to obtain a suitable protein or cell concentration in the enzyme reaction is not directly linked to the repair incisions because further dilutions of the crude protein extract occur, but is a useful indicator of relative amount of activity. Protein concentration or cell density in the final extract Essential Protein concentration directly affects the rate of repair incisions, so this information should be reported in articles. However, extracts from single-cell suspensions (e.g., blood samples or cell cultures) can be standardized to the same number of cells before the extraction of protein; the cell density in the final repair extract will then be equivalent to the dilution of the same starting cell number. Extraction buffer composition Desirable This is not likely to affect the repair activity as repair inhibitors are avoided in the buffer. Incubation buffer composition Essential Essential cofactors and buffer content may affect the activity of the repair enzymes. Optimization of enzyme concentration and duration of incubation Desirable See same item under section 4A. Volume of extract added to the gel Desirable Relevant for those who wish to repeat the experiment. Duration of incubation Essential See same item under section 4A. Temperature of incubation Essential See same item under section 4A. Mode of incubation Desirable See same item under section 4A. Type of enzyme used as positive assay controlterm Essential It is important to clarify the type of enzyme because they have different substrate specificities (e.g., Fpg and hOGG1 do not possess the same lesion specificity). Negative assay or background controls Essential It is crucial to demonstrate that the repair incisions are not just a result of nonspecific (background) damage to the DNA. 5 Alkaline treatment Composition Essential The composition of the solution can affect the conversion of alkali-labile sites to DNA strand breaks. The pH value is typically controlled by the amount of NaOH. Duration Essential Prolongation of the treatment can increase the conversion of alkali-labile sites to DNA strand breaks. Temperature Essential The temperature will affect the separation of DNA strands. 6 Alkaline electrophoresis Composition Essential The extent of DNA migration depends on the chemical composition. Voltage/cm over the slide support platform Essential The extent of DNA migration is directly proportional to the strength of the electrophoretic field. Duration Essential The extent of DNA migration is directly proportional to the duration of electrophoresis. The duration is restricted to avoided overlap between comets. Temperature Essential Electrophoresis at high temperature might induce DNA strand breaks and thereby increase the level of DNA migration. 7 Neutralization Composition Desirable No expected effect on DNA migration. 8 Staining and visualization Type of DNA dye Essential Dyes have different binding affinity to DNA and may therefore affect the calculation of primary comet assay descriptorsterm in the image analysis software. Concentration of dye Desirable Most likely does not affect the image analysis of the comets, but desirable informationterm for researchers who want to repeat the specific protocol. Time from staining until microscopy Desirable Certain dyes may require incubation to produce a good fluorescent signal. Microscope magnification Desirable For image analysis by software, the DNA migration differs between magnifications. Representative images of comets Desirable As the calculation of the %DNA in tail (or other descriptor) may be different between different image analysis systems, it is desirable to include images of comets and the level of DNA migration (e.g., as supplementary material or a citation to an earlier article with representative images, or by including images within figures). 9A Scoring and data analysis Type of primary comet assay descriptorterm Essential There are different ways to measure the level of DNA migration (i.e., %DNA in tail, tail length, tail moment and visual score). These primary comet assay descriptorsterm have different scales, which cannot be directly compared. Number of comets scored per gel and number of gels scored Essential Important because of low precision in the measurement of DNA in gels with few comets. Measure of the central value of comet scores (e.g., mean or median when image analysis systems have been used for analysis of DNA migration) Essential Using the mean versus median level of DNA migration might affect the estimate of DNA damage, depending on the distribution of comet scores. It is essential that authors clarify that mean/median values from comet distributions come from independent observations (i.e., different animals or humans, or cell culture experiments carried out on different days). Type of software for image analysis Essential Different software may have different algorithms for calculating primary comet assay descriptorsterm. Calibration Desirable The primary comet assay descriptorterm is a relative value (e.g., %DNA in the comet tail). Transformation to lesions per nucleotide or unaltered nucleobase pair is desirable for ease of comparisons between studies, although it does not affect the quality of the comet assayterm analysis. 9B Calculation of enzyme-sensitive sitesterm and DNA repair activityterm·def Calculation of enzyme-sensitive sitesterm Essential Results for the enzyme-modified comet assayterm should be reported as the net increase (i.e., enzyme-treatment with the ‘no enzyme’ level of DNA strand breaks subtracted). Calculation of DNA repair activityterm·def Essential Results for DNA repair activityterm·def should be reported as the net incisions (i.e., repair extract treatment with the background level of DNA migration subtracted). 9C Statistical analysis of results Essential The statistical analysis should conform to standard practice for parametric, nonparametric or logistic regression, depending upon the study design. a Information on each comet assayterm step is classified as either ‘desirable’ or ‘essential’ information based on a threshold of ≥75% agreement between the authors of this Consensus Statement. Methods The MIRCAterm guidelines have been crafted by members of hCOMET COST Action with the goal of improving the analysis and reporting of comet assayterm results ( http://www.hcomet.eu/ ). The authors are comet assayterm experts with a minimum of eight years of experience with these assays (15 of the authors have >20 years of relevant experience). We have used an internet-based questionnaire to sample opinions from the authors concerning the importance of reporting details (Table 1 ; Supplementary Data ). Each piece of information was graded by the authors as ‘essential’, ‘desirable’ or ‘not important’, and a threshold of ≥75% congruence was used for the classifications. If the number of ‘essential informationterm’ replies did not reach the threshold of 75% agreement, the ‘essential informationterm’ and ‘desirable informationterm’ replies were combined, and the piece of information was classified as ‘desirable informationterm’ if ≥75% authors agreed it was either ‘essential’ or ‘desirable’. In Table 1 , we include explanatory notes for each recommendation. Specific MIRCAterm recommendations for each step of the comet assayterm Step 1A: Isolation of cells and preparation of single-cell suspensions As the comet assayterm uses suspensions of single cells, specimens that are not obtained as single cells must be treated mechanically or enzymatically to disrupt the attachment of cells to an extracellular matrix or to each other. The homogenization of tissues by mechanical disruption may itself cause DNA damage, whereas the enzymatic digestion of tissue may lead to the removal of DNA lesions due to the activity of endogenous DNA repair enzymes, or increase DNA damage levels by releasing nucleases from the cells. The composition of the homogenization buffer is ‘desirable’ information, particularly in regard to the components that are necessary for preserving DNA integrity (e.g., ethylenediaminetetraacetic acid) 24 , 25 . A description of the procedure for the isolation of single-cell suspensions, including homogenization of the tissue, is considered to be ‘desirable’ information to report in articles. Blood samples are often used in human biomonitoring studies. Because blood represents a heterogeneous population of cells, detailed information on the cell population is ‘essential’ in publications. The text should precisely describe whether the specimens are whole blood (including red blood cells), leukocytes, mononuclear blood cells or a subset of cells (e.g., lymphocytes). It can also be helpful to include information on the procedure for venipuncture, type of anticoagulant and subsequent method of cell isolation (i.e., centrifugation and washing steps) for those repeating the experiment, so this is classified as ‘desirable’ information. Information on the storage conditions of the cells or tissues if they are cryopreserved, as well as the freezing method (e.g., snap freezing on dry ice or in liquid nitrogen, or a slow freezing procedure) and thawing procedure, is considered ‘essential’ information to report, as these processes have been shown to affect the basal level of DNA migration 26 . Step 1B: Preparation of substrate cells for the in vitro DNA repair assay Any eukaryotic cell type can be used as a substrate cell for the in vitro DNA repair assay, and the cell type and density are ‘desirable’ information to report. It is ‘essential’ to report the method and type of genotoxic compound used to induce DNA lesions in the substrate cells and subsequent cell storage conditions, whereas the total level of DNA lesions that can be detected in the substrate cells (e.g., the number of formamidopyrimidine DNA glycosylase-sensitive sites in cells treated with Ro19-8022 plus light) is considered to be ‘desirable’ information only. Step 1C: Assay controlsterm·def In this article, assay controlsterm·def refer to samples that are included in every comet assayterm experiment; these are sometimes called reference standards, internal controls or technical controls 16 , 27 . The assay controlsterm·def are typically cryopreserved aliquots from a single batch of cells that have been exposed to a DNA strand-breaking agent (e.g., ionizing radiation, hydrogen peroxide or methyl methanesulfonate) or a treatment that causes a specific type of DNA lesion (e.g., the photosensitizer Ro19-8022 plus light, or potassium bromate treatment, to induce DNA oxidation). There are different assay controlsterm·def for the standard alkaline comet assayterm and the enzyme-modified comet assayterm. The compound used for the enzyme-modified assay should not generate DNA strand breaks, as these decrease the dynamic range of the enzyme-sensitive sitesterm. In biomonitoring studies, as well as cross-sectional, interventional and clinical studies, unexposed cells can be used as assay controlsterm·def. It is ‘essential’ to report levels of damage in assay controlsterm·def and assay variation (standard deviation) in both the standard and enzyme-modified comet assayterm. The in vitro DNA repair assay uses internal experimental controls, which are also used in the calculation of the repair activity, rather than assay controlsterm·def per se. It is ‘essential’ information to report the level of DNA repair incisions in nucleoids from (i) non-exposed substrate cells incubated with reaction buffer, to determine the basal level of DNA damage in the substrate DNA (i.e., the ‘background control’); (ii) exposed cells incubated with the reaction buffer, to reveal the level of any nonspecific DNA strand breaks or abasic sites resulting from the treatment with the damaging agent (i.e., the ‘treatment control’); (iii) non-exposed substrate cells incubated with protein extract from the sample, to check for nonspecific incision or cleavage activity (i.e., the ‘specificity control’); and (iv) exposed substrate cells incubated with lesion-specific enzyme, similar to assay controlsterm·def for the enzyme-modified comet assayterm (i.e., the ‘incubation reaction control’). Step 1D: Negative and positive controlsterm·def In this article, negative and positive controlsterm·def refer to the experimental groups, as described in the OECD guideline (TG 489) for the in vivo comet assayterm in animal tissues 18 . Thus, negative and positive controlsterm·def pertain to the whole experiment. A positive control refers to a direct- or indirect-acting genotoxic compound that produces DNA strand breaks or enzyme-sensitive sitesterm detected with the comet assayterm. For the enzyme-modified comet assayterm, there is no list of positive controls available that corresponds to the compounds the OECD lists as positive controls for the alkaline comet assayterm (for inducing DNA strand breaks) in specific animal tissues. In addition, a positive control does not exist for human biomonitoring studies, as healthy people cannot deliberately be exposed to a genotoxic agent. Positive controls are already considered mandatory in cell culture experiments in genetic toxicology and will de facto be ‘essential’ information in articles using the comet assayterm. In animal studies, however, for practical purposes comet data on assay controlsterm·def (i.e., samples mentioned in the section above entitled ‘Step 1C, Assay Controlsterm·def’) are sufficient, whereas results from true negative and positive controlsterm·def are considered to be ‘desirable’ information only. Step 2: Embedding of the cells in agarose The comet assayterm was originally developed using three layers of agarose on glass slides, with the middle layer containing the cells. However, the top layer of agarose is not necessary, and certain procedures do not use a bottom layer (e.g., Gelbond film assays). It is ‘desirable’ to report the type of slides and size (i.e., surface area) of the gels, as the proportion of the cells near the edge of the gel increases as the gel size decreases and the DNA in nucleoids at the edge of the gel may migrate differently from that in nucleoids toward the center of the gel 22 . The final concentration of agarose (with the cells embedded therein) is ‘essential’ to report, as the migration of DNA depends on the density of the gel. Step 3: Lysis of the cells There are several procedures for lysing cells in the comet assayterm. Information about the composition of the lysis solution is ‘essential’. An extra enzyme incubation step (e.g., with proteinase K) may be required for certain types of cells, and details of the incubation should also be reported as ‘essential’ information. Some reports suggest that the duration of lysis may affect the stability of certain types of DNA lesions 28 – 30 , so the duration of the lysis and temperature of the lysis solution are ‘desirable’ information to report. Step 4A: Repair enzyme treatment in the enzyme-modified comet assayterm As the lysis solution may inhibit the activity of the repair enzyme, it is ‘desirable’ to establish whether a washing step was performed between the lysis step and enzyme treatment (i.e., composition of the washing buffer, number of washings and duration). It is ‘essential’ to relay information about the source of repair enzymes, as enzymes from different manufacturers have been shown to differ in both their activity and their specificity towards nucleobase lesions 16 . In most comet studies, titration curve experiments are performed to identify optimal conditions for the enzyme treatment 31 ; however, the results of enzyme titration curves are rarely reported and are classified here as ‘desirable’ information (reference to a previous study could be made instead), although we regard it as ‘essential’ to report the duration and temperature of the treatment. The concentration of enzyme applied to the gel is ‘essential’ information; it is preferable to report the concentration in enzymic units (U/ml), although the protein concentration (mg/ml) is also useful. The type of incubation unit (e.g., incubator, slide moat or 12-gel system) and mode of incubation are ‘desirable’ information to report. It should be stated whether the incubation was performed (i) in an enzyme bath or with a drop and coverslip on the slide, (ii) in a standard 2-gel version, 12-gel or multiple-well system or (iii) in a humidified box in an incubator, on a heating plate or in a heated slide moat. Step 4B: Extract preparation and incubation for the in vitro DNA repair assay It is ‘desirable’ to report the number of cells or mass of tissue used to prepare the protein extracts, whereas it is ‘essential’ to report the final protein concentration of cell or tissue extracts that is added to the substrate DNA. The composition of the extraction buffer (‘desirable’ information) is less crucial than the composition of the incubation buffer (‘essential’ information), because the latter can affect the background level of DNA migration. In keeping with the information for the enzyme-modified comet assayterm, it is ‘desirable’ to report the results from titration experiments or reference previous studies from the same laboratory, where these have been performed. The volume of extract added to the gel-embedded substrate cells is ‘desirable’ information. It is ‘essential’ to report the duration and temperature of the incubation period because these affect the number of repair incisions. As for the enzyme-modified comet assayterm, the mode of incubation is ‘desirable’ information to report for the in vitro repair assay. Information on the identity of the enzyme used as the incubation reaction control (indicating the amount of DNA lesions in the substrate cells) and the composition of the buffer used for the background level of DNA repair incisions in unexposed substrate cells are ‘essential’, because they are key to demonstrating the reliability of the DNA repair incision activity. Step 5: Alkaline treatment The high-alkaline-pH solution disrupts the hydrogen bonding that holds the DNA strands together and also converts certain nucleobase lesions into DNA strand breaks. Thus, the duration of alkaline treatment can affect the level of DNA migration in the subsequent electrophoresis 32 – 34 . Specific information concerning the composition of the alkaline solution, pH, temperature and duration of the treatment are thus ‘essential’ information to report. Step 6: Electrophoresis The most important drivers of DNA migration are the duration of electrophoresis and the electrical potential (voltage drop across the electrophoresis tank platform) 35 . It is ‘essential’ that the composition of the electrophoresis buffer, strength of electrophoresis (voltage gradient (V/cm) over the electrophoresis tank platform) and duration of electrophoresis are reported. High temperature during electrophoresis may affect the DNA migration 36 ; thus, information about the temperature of the electrophoresis solution is ‘essential’, and this may be accompanied by descriptions of steps taken to keep the temperature constant (e.g., cooling the platform or circulating the buffer). Step 7: Neutralization This step involves removing excess alkaline solution from the slides to ensure efficient staining. It is ‘desirable’ to describe the composition of the neutralization solution. Step 8: Staining and visualization DNA-binding dyes have differing binding affinities to DNA and may therefore affect the calculation of the primary comet assay descriptorsterm in image analysis software differently 36 – 38 . The type of dye is ‘essential’ information, whereas the concentration is ‘desirable’ information, as is the time between staining and visualization. The intensity of the light from different types of microscope lamps differs. In addition, it varies due to the age of the lamp and the time it has been turned on during the scoring of comets. However, there is no standard procedure for measuring the intensity of light and correcting the level of DNA migration accordingly. Furthermore, the same comet may appear to have different levels of DNA migration at different microscope magnifications. Thus, it is ‘desirable’ to report the microscope magnification used for the scoring. It is ‘desirable’ to show representative images of comets (e.g., control with little or no migration, moderate and extensive DNA migration) alongside the reported levels of DNA migration. Step 9A: Scoring and data analysis This step requires the reporting of ‘essential’ information for the primary comet assay descriptorterm (e.g.. %DNA in tail, tail length, tail moment or visual score), the number of comets that are analyzed per sample and how the overall level of DNA migration is expressed (e.g., median or mean of comet scores). It is ‘not important’ to report the individual result of each comet in each gel; they are combined to calculate the overall damage level. As it cannot be ruled out that different image analysis software packages may have different ways to calculate the primary comet assayterm predictor, it is ‘essential’ to report the software used (software name, manufacturer, version). All primary descriptors share the limitation that it is necessary to have expertise in the comet assayterm to understand what they mean, whereas if a calibration curve is created (using ionizing radiation, which induces breaks at a known frequency), results can be converted to relative lesion frequency compared to unaltered nucleotides or nucleobase pairs; such data are unequivocal and convey information that all researchers can understand 39 . The procedure to obtain a calibration curve, using ionizing radiation, and convert DNA migration levels to the lesion frequency relative to unaltered nucleotides or nucleobase pairs has been reported previously 40 . Thus, reporting the results as levels of lesions per 10 9 unaltered nucleotides or 10 6 nucleobase pairs is ‘desirable’. Step 9B: Calculation of enzyme-sensitive sitesterm or net number of incisions in substrate DNA for the in vitro DNA repair assay The incubation with DNA repair enzymes increases the level of DNA migration, as both the basal level of DNA strand breaks and enzyme-specific lesions contribute to the total number of DNA strand breaks. As the DNA migration that is attributed to the basal level of DNA damage and enzyme-specific lesions may originate from different mechanisms of genotoxicity, it is insufficient to report only the total level of DNA damage after enzyme treatment. Instead, it is ‘essential’ to report the genotoxicity as enzyme-sensitive sitesterm, where the basal level of DNA migration has been subtracted from the DNA migration in the enzyme-treated slides. As the in vitro DNA repair assay uses the same substrate cells with all cell or tissue extract samples, it is unnecessary to have concurrent background and treatment controls for each sample in the same experiment (i.e., assay run). There might be more than one substrate (e.g., when analyzing both base- and nucleotide-excision repair activity), and therefore separate controls for each type of DNA repair assay are needed. It is ‘essential’ to report the net incisions by the repair extract in the substrate DNA. Step 9C: Statistical analysis of the results Statistical analyses are ‘essential’. The type of statistical analysis depends on the design of the study and follows the general assumptions in statistical testing in genetic toxicology and biomonitoring 41 , 42 . Conclusion All articles that include results from comet assaysterm should have a clear description of the experimental protocol. In principle, they should contain as much information about the comet assayterm procedure as possible, but some details are more important than others. Most journals with a word count limitation have supplementary sections where the details of the procedure can be described. However, if this is not possible, authors should cite articles with a detailed procedure (preferably those that are open access or freely available). Terms such as ‘modified from’ and ‘adapted from’ should be avoided, unless the specific modifications are defined. The MIRCAterm recommendations represent a standardized reporting checklist for the description of comet assayterm procedures and results. However, this is not a guide to best-practice procedures for the assay. The MIRCAterm recommendations provide an important tool to aid researchers, reviewers and editors in ensuring that the comet assayterm is performed rigorously and reported comprehensively. Taken together, these will increase the quality and impact of comet assayterm results in scientific studies. Supplementary information Supplementary Table 1 Supplementary Table 1. Author contributions P.M., A.A. and S.A.S.L. organized the online questionnaire and drafted the paper. All authors answered the questionnaire and read and approved the final manuscript. S.A.S.L. and A.A. designed the figure. Competing interests The authors declare no competing interests. Footnotes Supplementary information is available for this paper at 10.1038/s41596-020-0398-1.