[Histonet] Tissue processing for laser microdissection and RNA isolation?

[Rene J Buesa]

If you fix adequately (no NBF) you can go with 1
René J.

From: Mikael Niku <mikael.niku <@t> helsinki.fi>
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Wednesday, February 13, 2013 8:15 AM
Subject: [Histonet] Tissue processing for laser microdissection and RNA isolation?

Hello!

May I ask for your recommendations for tissue processing methods for laser microdissection and subsequent RNA isolation?
I can think of at least the following protocols, each with significant drawbacks (and questions):

1) Traditional FFPE sections:
+ easy handling
+ RNA is safe (but see below)
+ good morphology
- RNA is fixed too, so yields are low and only small fragments retrieved
Here, I'm pretty happy with Qiagen RNEasy FFPE kit - any other suggestions, maybe cheaper?

2) Traditional cryosections:
+ fairly good morphology
+ good yields, good quality RNA if everything goes well
- RNA is easily destroyed
- difficult to handle small samples without melting & destroying RNA
I haven't been very succesful with this option.

3) RNALater -> cryosections:
+ RNA is safe
+ good RNA yields, good quality RNA
- poor morphology
- difficult to section
We have problems making the tissues actually freeze for good sectioning - any tricks or tips here?

4) RNALater -> paraffin sections?
I haven't tried this yet, but should be doable through ethanol etc. Found some references claiming that the RNA quality is poor.

5) New commercial innovations like Qiagen/Prenalytix Paxgene Tissue kit, claiming to achieve both RNA stabilization and good morphology.
I haven't tried any of these yet. Pricing is the obvious drawback.

With best regards,
Mikael Niku, PhD
Department of Veterinary Biosciences
University of Helsinki


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