Hello,
I have been trying very hard, to no avail, to get RNA from nematodes using
the 'glass-bead' protocol found in WBG Nov '89 p.154. It is a standard
guanidine thiocyanate/organic extraction protocol.
The problem is that even after homogenization with glass beads as described
(with PCI (25:24:1) / 4M GITC / 0.5% N-laurylsarcosine /
2-mercaptoethanol), a second PCI extraction and the first precipitation
step with 1/10 vol. 3M sodium acetate + 2 vol. 100% ethanol, when I suspend
the pellet in DEPC-water the resultant solution always contains high
amounts of RNase activity - so there is no RNA to recover afterwards.
(Addition of even a small part of the suspended pellet to another RNA
sample destroys it.)
I have checked, re-checked and re-made every solution to the right pH and
DEPC-treated the water and sodium acetate (both of which I know are fine by
testing on other RNA samples) to no avail - there is still a large amount
of RNase immediately following precipitation. The phenol I am using is of
neutral pH, so there is no base present at any step to hydrolyze the RNA.
Has anyone else had a similar problem with this extraction method?
I would appreciate any help I can get - we've run out of ideas.
Morris Maduro
morris_maduro at biology.ualberta.ca
