International: +44 1440 730773Comet assay hints and tips
Using fluorescent dyes
The most widely used dyes for the comet assay are ethidium bromide, propidium iodide, SYBR Green and SYBR Gold. Any of these dyes can be used with the Comet Assay IV system. Regardless of the dye used, there are some important factors to take into account:
- You should aim to achieve a level of brightness whereby when the cells are visualised onscreen, you can still see detail in the head region of the cell. Saturating the video image results in a loss of information about the head region. If your images are saturating, then consider the use of a neutral density filter.
- You should take steps if you find the brightness of the cells diminishes under excitation. If your cells are fading, then they will not provide you with consistent results across a whole slide. Try using an anti-fading agent such as Vectashield from Vector Labs, or try reducing the intensity of the fluorescence by using a neutral density filter.
- When you are not using the system, ensure that if the lamp housing is to be left switched on that the path of light is blocked to prevent burning out the excitation filter block.
Cell scoring and measurement parameters
The International Workshop on Genotoxicity Test Procedures recommend the use of image analysis for comet single-cell gell electrophoresis assays, and give % tail DNA, tail length and tail moment as the recommended measurement parameters. The following excerpt is from the paper that resulted from the meeting held in conjunction with the Fourth International Comet Assay Workshop:
"DNA migration can be determined visually by the categorization of comets into different 'classes' of migration (Collins et al 1993) or by using an eyepiece micrometer to estimate image or tail length. However, image analysis is recommended, with the measurements of parameters such as the percentage of DNA in the tail (percent migrated DNA), tail length and tail moment (fraction of migrated DNA multiplied by some measure of tail length). Of these, tail moment and/or tail length measurements are the most commonly reported, but there is much to recommend the use of per cent DNA in tail, as this gives a clear indication of the appearance of the comets and, in addition, is linearly related to the DNA break frequency over a wide range of levels of damage. The approach or parameter used must be clearly defined and, if not typical, be justified."
A. Hartmann, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. Collins, A. Smith, G. Speit, V. Thybaud and R.R. Tice. Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis, Vol. 18, No. 1, 45-51, (2003)
These same recommendations were upheld at the International Workshop on Genotoxicity Test Procedures at San Francisco in 2005, with a recommendation that per cent DNA in tail be the most suitable measurement parameter.
Statistical analysis
As the comet assay is not yet a regulatory assay, there are no absolute guidelines on the reporting of results obtained. However, a number of useful papers on the subject can be found. We particularly recommend reading the following paper, the guidelines of which were used when designing the Spreadsheet Generator macro that is included with Comet Assay IV:
Stig Johan Wiklund and Eva Agurell. Aspects of design and statistical analysis in the Comet assay. Mutagenesis, Vol 18, No.2, 167-175, (2003)
Slide preparation
For those not using pre-prepared slides for their assays, a common problem is one whereby agarose does not properly adhere to the slide.
This problem is usually caused by washing being too vigourous or not letting the slides dry completely before staining. Suggested advice is to reduce the agitation during washing, and to allow slides to dry naturally rather than inducing or speeding up drying using a heat source.
Have you got any useful hints or tips?
If you have any advice that might help others performing the assay, please fill in the form below to let us know. We will be happy to share your experiences with others.