Isolation of Total RNA from just about anything

Winoto lab lab_winoto at maillink.berkeley.edu.
Mon Jun 27 01:46:59 EST 1994


RNase-All total RNA Isolation						Astar Winoto
										Paul W. Diaz
										Elicia Penuel
Make the following: 
1. Guanidium Solution

50 g of Guanidium Thiocyanate (Fluka grade)
0.5 g Sodium N-lauryl sarcosine (NOT SDS !)
0.83 ml of 3 M NAOAc pH 5.2
0.33 ml of Antifoam (optional)
Add water to near 100 ml, stir in a warm plate
pH to 7.0 with a few drops of 10 N NaOH (~ 160 µl). Be careful not to
overshoot!
Add 0.7 ml of Beta-mercapto-ethanol and bring total volume to 100 ml
Filter through a 0.45 µ filter unit
Solution should be clear and weigh ~1.1 to 1.2 g/ml

2. Equilibrated Phenol (see Maniatis or "Current Protocols")


Procedure

	1.	Mix one volume of phenol with one volume of Guanidinium solution.
		This solution is referred to as  RNase-ALL

	2.	Add up to 100 mg of tissue to 2.0 ml of RNase-ALL at RT 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 req'd).

	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 ppt overnight do it at 4
oC.

	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 of 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 EtOH ppt 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 specturm of
RNAs including 4S and 5S RNA species.






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