[Zbrafish] Re: RNA extraction from fixed tissue
(by amiller from fhcrc.org)
Fri Dec 16 13:30:13 EST 2011
I am piloting similar types of experiments and, instead of fixing the tissue and then trying to extract RNA, I have been crudely dissecting the animals into halves (directly after approved euthanization) - one half to look at my tissue of interest and the other to extract RNA - and preserving each for subsequent downstream applications. I keep the animals separated into two 96-well plates such that animal1 is in A1 of both plates, animal2 in A2, ... etc. I keep the tissues on ice, and for RNA extraction I place it directly into 10uL of Trizol.
So far I have been able to use each piece for its intended use (antibody staining / RNA extraction) with decent results (good enough antibody staining to see my tissue but not beautiful / decent yield of clean RNA but a bit of degradation). I am currently trying to perfect both the fixation conditions and RNA extractions in small volume - but I think eventually it will work perfectly.
So, an alternative. And I would love to hear about anyone that has been able to get good quality RNA out of a fixed tissue.
> 1. RNA extraction from fixed samples (hannah.rouse.09 from ucl.ac.uk)
> From: hannah.rouse.09 from ucl.ac.uk
> Date: December 15, 2011 4:49:03 AM PST
> To: bionet-organisms-zebrafish from moderators.isc.org
> Subject: [Zbrafish] RNA extraction from fixed samples
> I would like to do qPCR and a microarray from zebrafish brains, but I
> need to fix them before RNA extraction and purification. Has anyone
> done this, and what sort of yield did you get from it?
> Invitrogen has a nucleic acid isolation kit, which can purify from
> fixed samples that have been in paraffin, http://products.invitrogen.com/ivgn/product/AM1975
> and I am thinking of using this, without the deparaffinization step,
> but if anyone has any prior experience of this, or a better/different
> way of isolating the RNA, please let me know!!
> Many thanks,
Adam C. Miller
Division of Basic Sciences
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N, Mail Stop B2-152
Seattle, WA 98109-1024
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