i did acridine orange assay that result showed 99% of sperm no DNA damage but in comet assay more than 10% of Tail DNA observed, Why this much of variation and how to correlate? please explain me.
Acridine orange assay is used to detect autophagy and this dye crosses into lysosomes. Comet assay is use to detect cellular DNA damage and any dye like DAPI, ethidium bromide can be use for comet assay. If you want to correlate cellular autophagic response with DNA damage you can use these two.
Please check the following link:
http://www.cometassayindia.org/protocols.htm
Acridine orange is used to detect apoptotic cells as it can easily penetrate into apoptotic cells causing a bright fluorescence. But, comet assay is specifically used for DNA damage. Generally level of DNA damage increases with the progression of apoptosis.
Actually both the assays are used to detect the DNA damage, but in acridine orange assay we do not have to electrophorese the so we can get the patches of staining in cell itself as well as cells remain intact. Whereas, in case of COMET assay we have to lyse the cells so the DNA content come out and then it is applied to electrophorese. As a result of applying electrohporesis technique in COMET the broken part of DNA showed long tailing, whereas in acridine orange assay it is not possible.
You can correlate both assays, based on DNA damage you observed.
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