Table 1.
Reporting checklist for comet assay studies
| Step | Comet assay parameter | Reporting requirementa | 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 assay (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 assay 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 controls | ||
| Essential | Assay controls should always be included and reported in studies that do not have a positive control group. | ||
| 1D | Negative and positive controls | ||
| Desirable | Control groups are desirable (or even essential in certain cases). For most purposes, however (and especially in human biomonitoring), assay controls can replace negative and positive controls (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 assay, 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 control | 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 descriptors in the image analysis software. | |
| Concentration of dye | Desirable | Most likely does not affect the image analysis of the comets, but desirable information 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 descriptor | 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 descriptors 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 descriptors. | |
| Calibration | Desirable | The primary comet assay descriptor 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 assay analysis. | |
| 9B | Calculation of enzyme-sensitive sites and DNA repair activity | ||
| Calculation of enzyme-sensitive sites | Essential | Results for the enzyme-modified comet assay 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 activity | Essential | Results for DNA repair activity 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. | ||
aInformation on each comet assay step is classified as either ‘desirable’ or ‘essential’ information based on a threshold of ≥75% agreement between the authors of this Consensus Statement.