MSP for formalin-fixed and paraffin-embedded tissues - (Apr/26/2006 )
Hi, I am new to DNA methylation. Recently, we designed three sets of primers. one set of primers (u) anneal to the unmethylated DNA, another set of primer (m) anneal to the methylated DNA. The third one is the wild-type primer (w) which used as control anneal to the DNA without modification. For the cell lines U and M worked all right, but I can the PCR bands for w primer both in modified and unmodifed DNA. I guess the CT conversion efficiency is not very good. I may have a wrong opinion about this. I think it does not matter because it's hard to get 100% CT conversion and maybe a little amount of unmodified DNA there may result in a visible band. So I just go ahead.
Next I'd like to see if they worked all right for formalin-fixed and paraffin-embedded tissues. To my surprise, there is no band for U and M primers, but I can still get the bands for W primers. It seems that in this kind of tissue the CT conversion efficiency is even lower than that in the cell lines.
I have seen that "Methylation specific PCR is commonly used to determine promoter methylation. However, it cannot be used with formalin-fixed samples because the DNA extracted from these samples contains formalin-induced DNA/DNA or DNA/protein crosslinks that hinder chemical modification of DNA by sodium bisulfite, an essential step in the methylation specific PCR analysis."
But I still think it should work. I am thinking to improve the CT converstion effeciency. Does anybody has any experience about this kind of tissue? I used the EZ DNA methylation kit, how can I improve it?
My PCR condition is 35 cycles of 94 for 45sec, 62 for 30sec and 72 for 30sec. The PCR bands are about 200bp.
Thank you for your reply! Thank you very much!
the reason for inefficient conversion is that during bisulfite the DNA has not denatured properley and this will be challenging in fixed tissue samples. I would suggest using a higher conc of NaOH at the denaturation steps prior to addition of bisulfite and maybe incubate the samples at 100C for 5 minutes as well, hopefully this would be enough to not only reverse the crosslinks but to denature the DNA as well. I should say that such a process will also degrade the DNA meaning there will be not much around to amplify after conversion
Thanks a lot! I will try that.