Comet Assay
Automation of Quantitative DNA Fragmentation Analysis Based on Single Cells
The following customizations were most favored by our clients:
- Automated detection of objects in single-cell gel electrophoresis samples.
- Integrated background correction to address uneven image backgrounds.
- Support of multi-gel samples and multiple exposure times for enhanced accuracy.
- Option to calculate key parameters, including tail moment, Olive tail moment, %DNA in the tail, and head/tail dimensions.
- Managing highly damaged cells, known as "Hedgehog" comets.
- Comprehensive display of an object gallery together with all parameters obtained.
The Comet assay, or single-cell gel electrophoresis assay, is a technique used to measure DNA fragmentation in individual cells. It is commonly employed to assess DNA damage resulting from exposure to chemicals or radiation. The method involves analyzing images of DNA migration patterns in an agarose gel. The extent of DNA fragmentation determines the migration distance, creating a structure resembling a comet's tail. Quantification of the damage is based on measurements of the 'head' (representing the intact cell) and the 'tail' (indicating the migrated DNA fragments).
With guidance from our application specialists, numerous users have effectively leveraged the Metafer Platform Software to design and validate workflows for detecting cells in single-cell gel electrophoresis preparations and extracting critical parameters for the Comet assay. In this scenario, Metafer's tools can be configured to present a gallery of detected objects alongside key evaluation metrics such as tail moment, Olive tail moment, %DNA in the tail, and tail and head dimensions. Each object can be easily relocated with a single mouse click. The machine-generated data can be summarized in intuitive histograms and scatter plots, enabling experts to review and interpret the results effortlessly. For a better standardization of measurement, the background conditions in the images can be measured, and the results can be corrected for uneven backgrounds. Objects are detected based on user-defined morphological criteria and thresholds between cells and background.
In general, there are no specific requirements for preparing the gels. However, it is important to ensure that the material used does not produce autofluorescence, as this could interfere with image acquisition. Additionally, creating gels that are as thin as possible is recommended, as this facilitates the definition of optimal automatic focusing parameters. There are also Metafer users who successfully use dried gels for the Comet assay.
In principle, any fluorescent dye that specifically binds to DNA is suitable for staining. In the simplest case, even DAPI can be used.
For digitization with Metafer, specimens are secured in metal frames, allowing for easy exchange without the need to handle individual slides. For specialized applications, Metafer can also accommodate frames designed for preparations that deviate from standard dimensions. For instance, frames designed for the Comet assay can hold larger preparations containing multiple gels.
While it is technically possible, manually overwriting the evaluation is not recommended. Establishing a routine for the Comet Assay involves defining a standardized evaluation process for your preparations, which serves as the foundation for validating your workflow. Manually modifying this evaluation would deviate from these established conditions, particularly since accurately assessing the degree of damage to a cell manually can be very challenging. If the results do not align with your expectations, it is better to adjust the underlying parameters instead. However, keep in mind that doing so may require revalidating your workflow.
If Metafer unexpectedly detects objects that you would typically exclude from the evaluation (such as artifacts), you can easily remove these during the review process. This is conveniently done while examining the gallery after the scan.
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MetaSystems offers Customization Packages for application workflows that have been successfully implemented for customer labs using standard Metafer platform functionality. It is expected that they can be implemented for other customer labs using similar workflows and slide preparation procedures. If a Customization Package is purchased, MetaSystems product specialists will – based on their experience from other similar application cases - support the customer lab in adapting the Metafer software configuration to their needs. The performance of the solution will depend on the quality of the customer slides and the expertise of the users, MetaSystems cannot specify or guarantee any performance parameters. The validation of the solution for clinical use is the sole responsibility of the customer lab.