RNA isolation

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Fri Dec 10 16:43:21 EST 1993


> We have some important blood samples at -80C: about 10 ml whole blood, in a rather strange
> solution:
> 
> 1.8 M guanidine
> 5.8 mM Na citrate
> 20 mM B-ME
> 0.12 % lauryl sarcosine
> 
> Our first attempts to get RNA out failed: we tried adding guanidine to 4M and CsCl to 2.37 M,
> then spinning it through a CsCl step: got nothing!!!!!!!
> 
> Any ideas how we can get enough RNA for RT-PCR out of these frozen samples?
> 
> Could you answer by E-mail to young at cmu.unige.ch
> 
> Michele Young
> 
It seems to me that isolation of mRNA from those samples might be
amenable to an acid phenol extraction method (Chomcynski, P. and
Sacchi, N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal.Biochem. 162:156-159,
1987).  All of the reagents already in your sample are present in the
guanidinium isothiocyanate solution used in this procedure.  An
additional advantage is that you do not need to use cesium chloride. 
The method, as we use it in our laboratory is as follows:

 I. RNA PREPARATION (You should first adjust the contrations of the
components in your cells lysate to be the same as solution D in the
above reference.

A.     Thaw cells (or lysed cells) on ice.

B.     Vortex at 4`C for 10 minutes (if you have less than 1 
million cells than you need to add 1ul of tRNA to cells 
before processing).

C.     Add 1/10 volume of 2M NaAc pH4, vortex.

D.     Add 1 volume of H2O saturated phenol, vortex.

E.     Add 2/10 volume of chloroform-IAA, vortex 30 seconds.  
Put on ice for atleast 1 hour vortexing every 20 minutes.

F.     Centrifuge at 12,000rpm* at 4`C for 20 minutes.

G.     Chill on ice for 20 minutes (this is to ensure that 
the interphase has settled).

H.     Harvest the aqueous phase (keep away from the 
interphase).

I.     Add an equal volume of isoproponol, vortex briefly.

J.     Precipitate at -70`C for atleast 90 minutes (I 
usually do this overnight ).

K.     Thaw on ice.

L.     Centrifuge at full speed* for 30 minutes.

M.     Wash  with 80% Etoh for 10 minutes at 4`C, full 
speed*.  Do this 2 times.

N.     Wash with 95% Etoh for 10 minutes at 4`C, full 
speed*. 

O.     Dry pellet.

P.     Dissolve pellet in TE pH7.5 (RULE:  1 million cells 
per 4ul of TE).


*these speeds are for a microcentrifuge
NOTE:  all tips and tubes should be autoclaved and all 
solutions should be made up in depc-treated h2o.

This will give you RNA that is clean enough for most routine uses,
including PCR and Northerns.

The main problem I see with the storage solution that your cells are in
may have allowed RNases to chew things up.  We routinely store our
cells in 4M Guanidinium isothiocyanate at -80 and found no degradation
as detected Northern blots for at least 3 months.

Good Luck
-jim-

==============================================================================
James F. George, Ph.D.              "Science is simply common sense at its
Department of Surgery                best -- that is, rigidly accurate in
University of Alabama at Birmingham  observation, and merciless to fallacy
205-934-4261 voice                   in logic"   --Thomas Huxley
txpljfg at uabcvsr.cvsr.uab.edu
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