I am looking for a way to detect DNA damage in rat blood. I expect the DNA damage to be mild, so sensitivity surely is an issue. Till now, I have consulted several people on what would be the best way to do this, but I get a different answer every time. At this point, several assays were suggested; gamma-H2AX, Comet, TUNEL, 8-OHdG and DNA fragmentation, but I am really not sure what to use and which one would be feasible to perform within a relatively short amount of time. Also literature does not give me any clear clue on the optimal test. I hope someone is willing to advise me on what to use (and especially why) so I can make a well-informed decision. I would me really grateful.
Thanks in advace!
Dear Lori,
What kind stress are you applying to induce DNA damage ? IR, chemical i.v., physical stress, high fat diet,...? Depended on that information, there might be more methods. e.g. urinary excretion of 8-oxodG as a surrogate marker or additional marker
But what I can tell you, is that gH2AX is an extremely sensitive marker, even psychological stress has an impact on lymphocyte gH2AX. If you have access to a laser scanning microscope go for it. Our lab just recently published a paper on that method in blood lymphocytes. http://www.nature.com/articles/srep22682
If you don't have access to a LSM, a collaboration might also be possible with the authors of the aforementioned paper.
all the best :)
Thanks a lot for your helpful answers. The stress we are planning to use is a light therapy with blue light, a bit higher in the spectrum than UV, but we only expect mild damage, so we will definitely try the gH2AX staining. We do have a proper microscope, and I am currently trying the method described by Olivier in 1 drop of blood, which could be especially good for rat experiments. So far the protocol seems to work fine and we have the proper microscope, so I think it will work. In addition we will measure 8-oxodG in urine (the levels in plasma are too low for our method). Thanks again!
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