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Author Topic: RNA from harsh environments  (Read 2796 times)

Offline Ylide

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RNA from harsh environments
« on: 18/06/2004 04:04:34 »
So my project for this summer is to try and recover RNA from formalin-fixed paraffin-embedded prostate tissue.  A quick summary of the paper is that the various HOXC genes are expressed in healthy cells surrounding cancerous prostate cells but not in any cell in a prostate unaffected by cancer.  Since there are multiple HOXC genes and there is no decent antibody to distinguish between them, we are relgated to using RNA.  (which we reverse transcribe and then run through PCR)  

So, my question is, do any of you have experience with recovering RNA from tissue that has been fixed with formalin and embedded in paraffin.  (a common histological technique)  We need to increase our number of cases and this is the only realistic way.  I've read protocols from people who have managed to do with with breast cancer cells and bladder cells, but not specifically prostate.  Any words of advice from you researchy types?



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Offline chris

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Re: RNA from harsh environments
« Reply #1 on: 18/06/2004 08:01:23 »
Hi Jason

we're extracting RNA from paraffin blocks here. This is breast, not prostate, although prostate is next on the list of tissues to tackle.

One way to do this is to cut sections through the tissue, mount one section on a slide so you can stain it and look at the morphology (to confirm what you have processed, and then extract RNA / DNA from the adjacent section. you could consider a proprietary RNA-extraction medium such as TRIZOL (which I'm using).

A major problem with paraffin-embedded tissue is contamination. The overflow paraffin from the embedding machine is recycled many times and could pick up spurious RNA.

Good luck

chris

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Offline Ylide

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Re: RNA from harsh environments
« Reply #2 on: 18/06/2004 14:58:12 »
We're using TRIzol as the extraction agent, I was more looking into ways to improve RNA yields from the tissue.  How old/new is the tissue you're working with?  Some of the cases we get were fixed in formalin for upwards of 6 days before embedding and are 1-2 years old, which I believe may be the problem.  We're trying some new runs next week with freshly fixed rat tissue to compare results with.  

Thanks for the tip about the paraffin embedding machine, I'll mention that to the histologists.



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Offline Ylide

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Re: RNA from harsh environments
« Reply #3 on: 02/07/2004 05:31:25 »
Just to update you, Chris, we've been successful in getting B-actin RNA from prostate tissue that's been fixed for a week in formalin, embedded, and stuck in archive for up to a year.  I double checked with the histolgists and they change out the paraffin in the embedder regularly.  

How are you cutting and processing the breast tissue?  I'd be interested in seeing your protocol, as my primary goal for the summer is to find the easiest, cheapest, most efficient method to go from block to PCR.  I can email you if you want, I have a lot of information about this I'd like to share.  



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Re: RNA from harsh environments
« Reply #3 on: 02/07/2004 05:31:25 »

 

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