What are the possible reasons of false-positive results of comet assay. Theoretically the DNA of control group is not impaired, but most of the cells is positive.
Comet assays are sensitive to anything that causes DNA damage. Even exposing cells to fluorescent light can have an effect (do expts in subdued lighting, put silver foil round prep tubes etc). Issues such as does the donor smoke, medical conditions, age etc also may have bearing. There will always be some low level of DNA damage and repair occurring all the time. The issue may be one of processing, or getting the appropriate matched controls.
Before answer this question, I need more information of your experiment, like which cell did you use? how was cell growth before used it? were all tools clean (without DNase)? did running buffer re-use too mush time?
Maybe you can check these points.
Perhaps unwanted damage has occurred during sample preparation. So check morphology of cells to ensure healthy appearance before sample preparation and handle cells gently to avoid physical damage. Keep cells on ice as much as possible and prepare cell samples immediately before combining with molten LMAgarose. If you had used a PBS solution, ensure it is calcium and magnesium free. Also work under yellow light.
what kind of cells are you using (animal / vegetable)? Try to keep the cells and solutions under ice, avoid sunlight (especially in vegetable cells). It may be that the solutions and buffers used have some genotoxic effect on cells.
It may be explained by the fact that every cell ,even those without any treatment exhibits a baseline damage level.
Cells that requires an isolation procedure: enzimatic, mechanical or both, usually become damaged and shows comets.
In other words, if you are sure that your cells have not been UV irradiated inadvertently , the comet procedure , was done under dim ligth, centrifugation times were respected , resuspension was gently , in that case the presence of comet in negative control samples should be related to extraction procedure or (in the case of blood, oral muccosa or any other cells obtained from humans) sample storage (temperature permisive for repair enzyme function) and transport ( shaking ) until bench procedure began.
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