Small RNA and Polysome isolation

LACYP at wsuvm1.csc.wsu.edu LACYP at wsuvm1.csc.wsu.edu
Mon Oct 17 12:18:52 EST 1994


Hi people,
 
My lab is working on isolating small RNA molecules (50-300 base size)
from hamster brains.  We're trying to enrich the preparations and
possibly get rid of some of the larger RNA molecules (ie mRNA, etc..)
 
I have been using Acid-Phenol extraction (with 10mM NaAcetate/EDTA
saturated phenol) and getting really good RNA yields ... tried the
Guanid. phenol method, but had too much trouble with the interface
being pulled up while pipeting off the sup. phase.
 
I have tried LiCl ppt ... its supposed to ppt larger molecules at
4 M concentrations (?)  over smaller ones, but didn't seem to work very
well.
 
Has anyone tried using the dT-mRNA columns to clean up preparations?
 
Any other ideas on small RNA isolation?
 
By the way, I don't have convenient access to an ultracentrifuge, so I'm
limited to Sorval speeds (<20,000 g) and also no swinging bucket rotors.
 
 
Also, RE Polysome Isolation... it seems like most procedures involve
sucrose gradient centrig.  ... are there any that don't?
(I haven't had a chance to drive over to library yet to check out
references to Methods in Enzymology that were previously posted).
 
Thanks for any help you might be able to provide.
 
Paul Lacy
USDA
lacyp at wsuvm1.csc.wsu.edu
lacyp at wsuaix.csc.wsu.edu
 
 



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