Re: RT-PCR on Paraffin sections

From:

Abizar Lakdawalla

Formalin fixed paraffin embedded tissue sections work quite well for in situ hybridization and related techniques..
Reasons:
Formalin inactivates RNAses therefore cells/tissues rich in RNAses often show better signals after formalin fixation instead of frozen/alcohol fixation.
Enzymes don't work in dehydrated conditions (in wax blocks) therefore RNA integrity should be preserved.

The procedure may need to be modified
Proteinase digestion is often necessary to "permeabilize" the tissue
Antigen retrieval in addition to proteinase digestion may also help
Washes may need to nemore stringent to decrease background.

Abizar
www.innogenex.com

James Mubiru wrote:

Hullo everybody
I have a question on using formalin fixed paraffin embeded tissue sections for In Situ RT-PCR studies. I have been told that almost all of the RNA is degrded by the tissue processing procedures that it is almost a waste of time. I have been told to instead try frozen sections. But I love the cell detail I see with paraffin sections such that I want to give paraffin sections a try. My question to you out there is that has anybody tried to do In Situ RT-PCR on paraffin sections and what their experience was. I have heard of a product called RNAlater from Ambion. Has anybody used it for paraffin sections and what was their experience with it.
James Mubiru

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