I am trying to do chromosomal breakage test for stress cytogenetics. I have used 50ng/ml and 100ng/ml concentrations of mitomycine c. But i didnt get any single breakage in any of the concentrations. Is there any another solution for this? I have also followed the attached paper for this protocol but cant find the result.
thanks in advance.
In my experience doing chromosomal breakage (about 8 years) there are a few places where this can go wrong. First are you doing breakage on blood as in the pdf? because on immortalized cell lines you harvest at 48 hours not 72 (the key is to estimate 2 replication cycles and harvest then). Because radials are transient, they don't occur until a certain timepoint and then radial-containing cells die so if you look too late you can miss them too. Also it is possible that your cells are just not breakage positive if they do not have a Fanconi anemia or HR defect. Have you run your protocol on a positive control to make sure the reagents and timing are working?
thank you Nichole owen. Yes i have done as pdf and also with another protocol. As you said may be the time of incubation is high should i try 24 hrs incubation? And i dont have positive sample yet so i tried on normal sample but as i know there should be some breakages in normal cells also as we used mitogen at higher concentration. Isn't it true?
Please refer to this publication
Article Clinical and Molecular Heterogeneity of RTEL1 Deficiency
Here we have performed chromosomal breakage studies. Hopefully it would help you.
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