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No amplification in PCR using gDNA extracted from formailin fixed paraffin embed


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#1 FFPE1

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Posted 07 September 2009 - 06:04 AM

Hi,

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!!

#2 frankfan

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Posted 07 September 2009 - 11:05 AM

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. :blink:

#3 FFPE1

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Posted 08 September 2009 - 01:27 AM

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. ;)



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......

#4 gebirgsziege

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Posted 08 September 2009 - 03:22 AM

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.
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