Questions on the RNA extraction

Peter S. Cooper cooper3 at niehs.nih.gov
Thu Dec 12 09:11:44 EST 1996


REGCHAK at USTHK.UST.HK wrote:
> 
> Hi all,
>      I did a total RNA extraction on plant tissue by using the Guanidinium
> Method as usual.  However, when I finished all the preciptation and washing
> step, I cann't dissolve all my suspected RNA pellet in the 400ul DEPC
> treated water even under 65oC for half an hour. I am sure that 170mg leaves
> should not cause such yield of RNA.  What's wrong in my RNA pellet?  And I
> don't whether too dry of the pellet will cause the loss of the yield?
> 
>      Thanks you for any suggested answer.
> 
> Regina

The total RNA pellet even from animal tissues can be difficult to
dissolve. I'm not sure which of the various guanidinium methods you are
using, but I understand that much polysaccharide material and other junk
co-precipitates with the RNA in the Chomczynski and Sacchi (TriReagent
a.k.a. TriZol) protocol -acid guanidium thiocyanate phenol chloroform
extraction. This can be a particular problem with plant RNA. You might
try precipitating RNA with LiCl instead of with alcohols. This should
not bring down the polysaccharides. If you are using the commercial
reagent mix such as Tri Reagent, I know Chomczynski et al. have modified
the protocol for such tissues. Their approach is to salt out the
polysaccharides and proteoglycans. They suggest adding 0.25 ml of
isopropanol and 0.25 ml of 1.2 M sodium citrate, 0.8 M NaCl- use
disodium citrate, no pH adjust- to the aqueous layer following phase
separation. (The volumes given are per ml of TriReagent used in the
intial extract.) I hope this helps.

Peter 
-- 
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Peter S. Cooper, Peptide Neurochemistry, Mail Stop C3-04
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709
Phone: (919) 541-3238
FAX:    (919) 541-0626
email: cooper3 at niehs.nih.gov
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