Help: RNA Extraction from Paraffin Embedded Tissue

Whistler, Toni taw6 at cdc.gov
Wed Mar 21 14:28:54 EST 2001


We have been doing this for a while and successfully. We have tried several
RNA kits and all seem to work. The important thing to remember is that the
cross-linking by the fixation agents tends to break up the RNA into small
fragments. Therefore you cannot use any size exclusion methodologies, the
other thing that needs to be considered because of the fragmentation is
product size in the RT-PCR is key. The literature says that one can get up
to 450 bp. I find it is dependent on the archival material, tissue etc. I
seem to have success with products at 250 bp and less - no matter what the
material.

Hope this helps.

Toni 

-----Original Message-----
From: ymwang at mcw.edu [mailto:ymwang at mcw.edu]
Sent: Wednesday, March 21, 2001 12:00 PM
To: methods at hgmp.mrc.ac.uk
Subject: Re: Help: RNA Extraction from Paraffin Embedded Tissue


I have a similar question, i.e. how to extract RNA from paraffin embedded
tissue
and was wondering if you have gotten any suggestions.
Thanks,
Youmin Wang


"Anthony C. Hilton" wrote:

> Hi,
>
> We are currently trying to extract RNA (specifically mRNA) from archived
> paraffin embedded (PE) renal tissue for subsequent use in a Northern blot
> assay.  I'm aware of the difficulty in doing this, especially as much of
the
> stability of the RNA in the sample is dependent upon the steps taken to
> prevent RNase activity prior to paraffin embedding.  We have tried a
number
> of extraction kits which, while they accept the shortfall of their
products
> in doing so, claim to be able to extract sufficient mRNA from PE tissue
for
> a RT-PCR step.
>
> Has anyone any experience of getting mRNA out of PE tissues and if so what
> method would you recommend.  Are there any steps in the RNA extraction
> process which can be successfully optimised to enhances chances of
success?
>
> Any comments welcome as we are running out of ideas and this is putting
our
> research 'on hold' at the moment.
>
> Thanks
>
> Anthony Hilton

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