[Histonet] RE: FFPE PCR

[Sarah Dysart]

I do the PK digestion at 55C overnight and get the best results

Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of M. Kap
Sent: Sunday, June 23, 2013 12:14 PM
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] FFPE PCR

ProtK digestion at 55 degrees C for half an hour will do fine

There is also a new FISH like technique to detect single RNA molecules in situ, can't remember the name atm. But it works with so called Z-probes and it's very specific and sensitive. Especially when you want to look at virus in tissue it would be quite intersting to see where the virus is situated.

You may even consider PAXgene fixation from now on, great histo/molecular combo fixation. 

Best regards,

Marcel Kap 

Verstuurd vanaf mijn iPad

Op 23 jun. 2013 om 19:05 heeft "histonet-request <@t> lists.utsouthwestern.edu" <histonet-request <@t> lists.utsouthwestern.edu> het volgende geschreven:

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>   1. PCR on paraffin sections (E. Wayne Johnson ???)
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> Message: 1
> Date: Mon, 24 Jun 2013 00:27:06 +0800
> From: "E. Wayne Johnson ???"  <ewj <@t> pigsqq.org>
> Subject: [Histonet] PCR on paraffin sections
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <51C721DA.70208 <@t> pigsqq.org>
> Content-Type: text/plain; charset=UTF-8; format=flowed
> 
> Hi.
> 
> We would like to start doing PCR on paraffin fixed tissues from pigs..
> 
> It has some appeal to us because we can see the lesions in the tissue
> and then we can go after the causative agent post-hoc.  It may be useful
> in cases where we have clear lesions on histopath but not the right 
> fresh tissues
> for PCR.  We are very limited on what we can do with IHC because
> of the extreme difficulty of procuring the primary Ab.  It's very
> simple to make PCR primers here.
> 
> We do have PCR up and running for all of the diseases that we
> are interested in.  I understand that the process is basically transferring
> the sections to a small tube instead of a slide, then dewax with a little
> xylene, and remove the xylene with alcohol, then digest the section
> to release the viral RNA.
> 
> Does anyone have any recommendations, advice, or experience with the 
> digestion
> step, and can suggest an appropriate enzyme mix for this digestion?
> 
> Yes I am considering in-situ hybridization but we have working PCR methods
> so that seems to be a simple logical step.
> 
> E. Wayne Johnson
> Enable Ag Tech
> Beijing.
> 
> 
> 
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> End of Histonet Digest, Vol 115, Issue 26
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