Positive control for bisulfite converted genomic DNA? - (Sep/13/2005 )
I posted here a couple of weeks ago, because I had some problems with my methylation specific PCR (no bands/smear in 1st and 2nd step of my nested PCR).
Obviously I still have problems. The universal PCR works now, however after all I get mixed results for the MSP and USP. Basically I see bands of the correct size everywhere. I know that this could be due to partial methylation, but from some of my samples I know that the promoter of my gene of interest is not methylated [at least I would assume that this gene in primary cells is unmethylated (they also have the mRNA and the functional protein, which the cancer cells both lack)].
And even if it would be partially methylated I would still say my results don't make sense, since the bands for the primary cells are even brighter than the ones of the cancer cells (especially in MSP).
So, I thought my primers don't work and tried some other ones. But they wouldn't work either. At this point I don’t know if this is due to my (mis-) designed primers or my template (the bisulfite converted gDNA). Are there any positive controls (like b-Actin in normal PCR) for bisulfite converted genomic DNA, preferrably comercially available? This way I could rule out that my template is the problem.
Alu-PCR confirmed that the integrity of my genomic DNA BEFORE bisulfite treatment (EZ-DNA Methylation Kit, Zymo Research) was ok.
Please let me know, if you know of any controls at this point or what else could be the problem. I really appreciate your help!
hazel, you may have saturated your PCR by performing too many cycles so what you are seeing is the plateau stage of the reaction, ie: excess template, you need to catch the reaction in the exponential phase and to do this you need to limit the number of cycles, both methylated and unmethylated templates will exist in all your samples but in different proportions.
try stopping your PCR after 10, 15 and twenty cycles and I would bet that you will see the differences in band intensity at one of these time points.
the best way to visualise this if you have the luxury, is to do qPCR.