RNA isolation

[Jim Owens]

In article <303gk6$ilq at nic.umass.edu> Bindu Chawla,
bindu at titan.ucs.umass.edu writes:
>Hi, I have been trying to isolate total RNA from styles of flowers. The
problem 
>is that one of the most abundant proteins in the style is RNAse and
therefore i 
>have been getting partially degraded RNA, I have been usung a buffer
which has 
>high concentration of urea and b-mercaptoethanol. Also I haven't been
using 
>DEPC water or baking my glassware. This seems to have no effect on leaf
RNA 
>isolation, though. Any suggestions would be greatly appreciated. 

There was a message from lab_winoto at maillink.berkeley.edu two-and-a-half
weeks ago that gave a method you might try.  I have not used this method
myself, but it is very similar to the one routinely used in my group with
good results.

Good luck,

Jim Owens

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_Guanidinium Solution_
			50 g of guanidinium thiocyanate (Fluka grade)
			0.5 g sodium N-lauryl sarcosine (NOT SDS!)
			0.83 ml of 3M NaOAc pH 5.2
			0.33 g of Antifoam (optional)
Add water to near 100 ml, stir on a warm plate
pH to 7.0 with a few drops of 10M NaOH (~160ul).  Be careful not to
overshoot!
Add 0.7 ml of beta-mercaptoethanol and bring volume to 100 ml.
Filter through a 0.45 um filter unit.
Solution should be clear and weigh ~1.1 to 1.2 g/ml

_equilibrated phenol_
(see Maniatis or "Current Protocols")

Procedure:
1	Mix one volume of phenol with one volume of Guanidinium solution.  This
solution is RNase-ALL.

2	Add up to 100 mg of tissue to 2.0 ml of RNase-ALL at room temperature
and immediately homogenize with a motor driven homogenizer.  The
homogenization is preferably done in a 10 ml round bottom polypropylene
centrifuge tube.

3	Add 1/10 volume of chloroform:isoamyl alcohol (24:1), vortex for 20
seconds and incubate on ice for 20 minutes.  This step is crucial; don't
skip it or change it!

4	Centrifuge at 10, 000xg for at least 15 minutes (longer than 20' is not
required).

5	*Transfer the upper phase to a fresh 10 ml tube and add one volume of
isopropanol.  Allow the RNA to precipitate for 1-2 hours at -20o C.  Do
not exceed this time frame.  If you wish to precipitate overnight, do it
at 4o C.  

6	Wash with 80% ethanol and resuspend the RNA pellet in 1.0 ml of
DEPC-treated water.  Store at -70o C.

*	If the upper phase is tinted or cloudy, transfer the upper phase to a
new tube containing 1.0 ml phenol (not RNase-ALL).  The phases will
immediately mix.  Add chloroform as above in step 3 and complete steps
4-6.

Note:  The RNA is now suitable for RNase protection, northern analysis
and reverse transcriptase reactions.  If you wish to isolate polyA RNA
then an ethanol precipitation must follow step six.  Use 1/8 volume 3M
NaOAc pH 6.0 as the counter ion.

Yields are very high and almost invariably contain the full spectrum of
RNAs, including 4S and 5S species.
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