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Bisulfite modification of DNA from paraffin embedded tissue - (May/16/2007 )

Hi,

I am doing a study on GpC methylation. The samples I've got here are paraffin embedded tissue. What I did was using xylene to de-wax the slice and using Qiagen DNA Kit to extract DNA. After that, I used Qiagen Epitect Bisulfite kit for modification. Nested PCR was used. I run my 2nd amplicon in 2.5% Agarose gel. but only positive control showed a specific band on it. blush.gif All my samples showed nothing. sad.gif My positive control was the modified DNA extracted from frozen tissue along side with my paraffin tissue. I heard that DNA from paraffin embedded tissues are fragile. DNA recovery are low in compare to frozen tissue after bisulfite modification.

Did anyone have this problem before? Any suggestion for me?

Thanks sad.gif

-kct787-

Hi my lab routinely perform bisulfite PCR on DNA isolated from FFPE material. A couple of suggestions are make sure your amplicon is small - anything over 400bp is going to be very very hard to amplify from FFPE, under 200bp should amplify in most samples in my experience. Try extending the length of proteinase K treatment - i think the qiagen kit only treats for like an hour or something, we usually leave overnight @ 55 degrees to get as much DNA out as possible. I have never used the epitect kit myself.

try reading this for some tips
http://www.ncbi.nlm.nih.gov/entrez/query.f...ed_ExternalLink

-frozenlyse-

kct787,

one thing you could try is to perform a third round of PCR, false positives can arise from doing this due to cross contamination, however you may have very little DNA to amplify with and maybe a third round could bring up the amplicon.

just one of many suggestions that could be useful to you.

Nick

-methylnick-

QUOTE (frozenlyse @ May 17 2007, 06:05 PM)
Hi my lab routinely perform bisulfite PCR on DNA isolated from FFPE material. A couple of suggestions are make sure your amplicon is small - anything over 400bp is going to be very very hard to amplify from FFPE, under 200bp should amplify in most samples in my experience. Try extending the length of proteinase K treatment - i think the qiagen kit only treats for like an hour or something, we usually leave overnight @ 55 degrees to get as much DNA out as possible. I have never used the epitect kit myself.

try reading this for some tips
http://www.ncbi.nlm.nih.gov/entrez/query.f...ed_ExternalLink


Hi frozenlyse,

Thanks for your reply. My first round pcr amplicon is about 280bp and 2nd round is about 100bp. I suspect DNA loss during DNA isolation from FFPE makes my pcr difficult. huh.gif How do you isolate DNA from FFPE material? Do you use commercial kit? What I do is using xylene and wash it with 100% and 70% ethanol.

Thanks blush.gif

-kct787-

QUOTE (methylnick @ May 17 2007, 07:36 PM)
kct787,

one thing you could try is to perform a third round of PCR, false positives can arise from doing this due to cross contamination, however you may have very little DNA to amplify with and maybe a third round could bring up the amplicon.

just one of many suggestions that could be useful to you.

Nick


Nick,

Thanks for your suggestion. 3rd round sounds nice. But im afraid those non-specific product might appear. I will have it a try to see if it is really because i got little DNA at the first place.

Thanks

-kct787-