RT-PCR in paraffin embedded tissue sections

Sujatha Byravan IAULCSR at MVS.OAC.UCLA.EDU
Thu Apr 28 19:51:00 EST 1994


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?
                                          Thanks,
                                          Sujatha Byravan



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