RT-PCR in paraffin embedded tissue sections

David M. Berman eyeball at netcom.com
Fri Apr 29 15:46:21 EST 1994


Sujatha Byravan (IAULCSR at MVS.OAC.UCLA.EDU) wrote:
: I am having problems with in situ RT-PCR using paraffin  embedded tissue
: sections. In brief this is what I have been doing so far:
:    Dewax,de paraffinize and then treat with protease. I used trypsin
:    at 0.05mg/ml at 370C for 10-30 mins. Then, DNase treat O/N at 37C in
:    DNase buffer. I then inactivate the DNase in 2X SSC at 70 C. The
:    tissue crumbles after this step when added to buffer. Today, I used
:    Pro K at 6ug/ml at 37C for 7, 10 and 13 mins. I also increased the Na
:    Cl [] to 100 mM during the DNase digestion so that the tissue is not
:   kept O/N in a hypotonic soln. Above this [] DNase activity drops
:   (according to Promega). I dont know if this will work. Any ideas onwhy
: the tissue is crumbling with this procedure?


Sometimes the type of slide used makes all the difference in the
world.  The ones that work the best for me when I have to boil the
tissue on the slide are Fisher's positively charged "Plus" slides.
They're pretty reasonably priced and work well forin situ and
immunohistochem.
Beyond that, you may be dewaxing or rehydrating too abruptly; Nuovo's
instructions are much more rapid thatn standard protocols.  Try at
least 2 minutes in 100, 95, 70, and 50% EtOH before progressing to
water.

Good luck

-- 
                                             eyeball at netcom.com



More information about the Methods mailing list