Extracting DNA from fixed tissue?

Chris Zarow zarow at molbio.usc.edu
Wed May 25 20:12:42 EST 1994


Greetings to all.

     I have been trying to extract genomic DNA from formalin
fixed human cerebellum. Since I am having no luck, I welcome your
insights and suggestions.
     I started out using a standard DNA extraction protocol
(Short Protocols in Molecular Biology) where the tissue (about 1
gram) is homogenized in 10 volumes of 100mM NaCl/10mM Tris pH 8.0/25
mM EDTA/ 0.5% SDS/ 0.1 mg/ml proteinase K.  (I am using a Tissue
Tearor mechanical homogenizer.)  The homogenate is incubated
overnight at 50 degrees C.  Following two phenol/chloroform
extractions, DNA from the aqueous phase is precipitated with ethanol.
     Although I have used this method with good results for
unfixed human cerebellum, I have obtained zero DNA from fixed
tissue.
     After the standard method failed, I consulted some protocols
which describe methods to run PCR on DNA from paraffin-embedded,
formalin fixed tissue. I proceeded from the premise that formalin
fixed tissue has not been processed like paraffin-embeded tissue
has, and therefore the DNA should be easier to extract. On the
assumption that the proteinase K might be being inhibited or
inactivated by the formalin, I tried washing the tissue overnight
in 1000 volumes of running water to remove the formalin.  Then I
added 0.1 mg/ml proteinase K to the homogenate every 2 hours, to
a final concentration of 0.5 mg/ml at 50 degrees C. Still no DNA.  I
also tried to extract DNA using a boiling method from one of the PCR
papers.  Still no DNA.
  
     One observation which might give someone some insight is
that the white interface following the phenol/chloroform
extraction is much thicker with the formalin tissue than with the
fresh tissue.  I can't help but think that the DNA is somehow
still complexed with protein and is trapped in the interface, but
I'm not sure how to get it out. 
  
     I would appreciate receiving any comments/ suggestions/
insights/ solutions to this problem.  Thank you.

Chris Zarow, Ph.D
University of Southern California
-- 
Chris Zarow, Ph.D
University of Southern California
Rancho Los Amigos Medical Center
zarow at molbio.usc.edu



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