RNA from fixed tissues.

b-butler at nimr.mrc.ac.uk b-butler at nimr.mrc.ac.uk
Mon Apr 24 08:54:38 EST 1995


In article <abrim-2004952309500001 at 129.105.41.196>, abrim at casbah.acns.nwu.edu (Abir Mukherjee) says:
>
>Has anyone ever tried to isolate RNA from paraformaldehyde fixed tissues ?
>Any suggestions? Experiences? Ideas?

Hi
I am trying to do this from mouse spleens and lymph nodes
At present I have had moderate success with a couple of published methods
References:
Stanta et al 1991 Biotechniques 11:25
Weizsacker et al 1991 Biochem.Biophis.Res.Comm. 174:176

I have slightly modified the system.

After fixing overnight in 4% Paraformaldehyde I always take the tissue to 70% Ethanol
30 min PBS
30 min Saline
30 min 50% Ethanol: 50% Saline
30 min 70% Ethanol: 30% Water
The tissue can now be kept at +4 C indefinitely

Blot dry and add 200ul of either buffer-

Stanta lysis buffer:-
	6mg/ml Proteinase K
	1M Guanidium Thiocyanate
	25mM 2-Mercaptoethanol
	0.5% Sarcosyl
	20mM TRIS
Incubate 1.5hours at 45 C.
Or

Weizsacker lysis buffer:-
	500ug/ml Proteinase K 
	20mM EDTA pH 8
	20mM TRIS pH 8
	2% SDS
Incubate 1.5hours at 60 C.
After incubation I add 300ul of our Standard Lysis Buffer(4M GuSCN; 25mM Sodium Citrate;.1M 2ME;.5%Sarcosyl)
Aspirate through a 19g needle and freeze tissue overnight.  The following day you can phenol:chloroform extract.

Hope this is of some use
Best 
Bob

If we knew what we were doing it wouldn't be research!





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