PCR of formalin fixed tissues

[slebos at amc.uva.nl]

In Article <24NOV94.08413405 at admin3>
chelack at admin3 writes:
>I am also trying to set up PCR methods for formalin fixed tissue sections
>and am concerned that the length of time the tissue was in formalin greatly affects the ability to amplify products.  In general I am deparaffinizing the
>sections followed by freeze/thaw fracturing of the tissue and lengthy
>proteinase K digestion then phenol chloroform extraction.  We are working
>on the identification of M. bovis in elk samples.  The samples we have are
>fixed for an unknown length of time and actually may vary in fixation time
>among the samples.  So far I am able to obtain product from 4/7 samples
>which leads me to believe that fixation time may be the culprit.  Does anyone
>else out there have any other suggestions regarding this, or any other secret
>tricks they wish to share?
>                        Regards
>                        BJC     Never worry about others trying to steal
>                                your ideas, if they are any good at all
>                                you will have to ram them down their throats.

We have been amplifying DNA from fixed tissues for quite some time. The best
protocol we have is actually very simple. It was described in Diagn. Mol.
Pathol. 1, 136-141 (1992). The concept is that you incubate the tissue in prot
K and a non-ionic detergent like Tween (in which Taq amplifies perfectly fine),
incubate for one night at 56C to reverse the formalin fixation, and use the
resulting supernatant directly for PCR. This has worked on over 90% of the
cases we tried.

Good Luck,
Rob Slebos

University of Amsterdam Medical Center
Dept. Pathology
Meibergdreef 9
1105AZ Amsterdam
The Netherlands
E-mail: slebos at amc.uva.nl