No amplification in PCR using gDNA extracted from formailin fixed paraffin embed - (Sep/07/2009 )
I have a problem that I'm hoping someone will be able to help me with!! I am extracting gDNA from tissue which is stored as formalin fixed and paraffin embedded. I have been using the qiagen DNEasy kit to do this (first dissolving the paraffin in xylene). After clean up my 260/280 ratio looks good and I have approx 3ng/ul DNA in most of my samples. However, when I go ahead with the PCR I am not getting any product using the gDNA from the formalin fixed paraffin embedded sections as template. My positive control amplifies great every time..... So I've no idea why I am getting a 260 reading for DNA when I cannot amplify from it. I have checked to see if there is something in these PCR reactions which are inhibiting the reaction. This is not the case as I checked by spiking these PCR reactions using my positive control and the PCR worked.
I am now running the gDNA (before PCR) on a gel to examine it. I have also designed all of my primers to amplify up products of </= 220bp to maximise the chance of amplifying products from formalin fixed paraffin embedded tissue (in the case of DNA fragmentation). I am also trying a few different methods of extracting the gDNA from the formalin fixed paraffin embedded tissue - I am boiling the sample and treating with proteinase k before clean up and I am also going to try phenol chloroform method.
Has anyone encountered this problem before? Or any solutions to the problem?
Thanks for any help you may have!!
gDNA you got is total genomic DNA, the amount (3ng/ul) is OK. When I worked with clinical samples, sometimes I only got 0.03ng/ul, but still can get amplification.I am wondering whether you should optimize PCR conditions first, different annealing temperatures, different primers, et al. Good luck.
frankfan on Sep 7 2009, 12:05 PM said:
Thanks for your help. I have optimised all the PCR conditions on gDNA from cell lines before I go ahead with the gDNA from the formalin fixed paraffin embedded sections so this should be fine......
have you tried serial dilutions of your gDNA (like 1:10, 1:100, 1:1000)? maybe you still have some inhibitors left which can be diluted out? And as frankfan pointed out very few DNA can be enough for your PCR.