Isolation of Total RNA from just about anything
lab_winoto at maillink.berkeley.edu.
Mon Jun 27 01:46:59 EST 1994
RNase-All total RNA Isolation Astar Winoto
Paul W. Diaz
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
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")
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
homogenize with a motor driven homogenizer. The homogenization is
preferably done in a 10 ml round bottom polypropylene centrifuge
3. Add 1/10 volume of chloroform: isoamyl alcohol (24:1), vortex for
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
5. *Transfer the upper phase to a fresh 10 ml tube and add one volume
isopropanol. Allow the RNA to precipitate for 1-2 hours at -20o C.
not exceed this time frame. If you wish to ppt overnight do it at 4
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
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.
More information about the Methods