Total RNA isolation from cell pellets
General considerations when working with RNA:
- wear gloves at all times
- use RNase-free tips and tubes for any samples
(autoclaved and not touched without gloves)
- use RNase-free solutions (either Molecular Grade Water [Mediatech in supply center]
or order DEPC-treated solutions from Ambion)
- Trizol contains denaturants and is hazardous (similar to working with Phenol)
1.
Add 2 ml Trizol (Invitrogen)(in 4C fridge) to 2.5 x 106 cells (frozen pellet or fresh cells).
2.
Homogenize with polytron or resuspend cell pellet using a 3 ml syringe and 21-guage
needle, 3 cycles through needle.
3.
Switch to a 22-gauge needle and process 3 times.
4.
Incubate homogenized samples for 5 min at room temp.
5.
Add 200l (1/10th volume) chloroform/isoamyl alcohol (49:1, v/v), cover tubes tightly
and shake vigorously for 15 sec.
6.
Incubate at room temp for 2 to 3 min.
7.
Centrifuge samples for 15 min at 12,000 x g at 4C
8.
Transfer supernatant to new microfuge tube.
9.
Add 10M LiCl and DEPC water to a final LiCl concentration of 2.5M to precipitate RNA.
10.
Incubate at -20C for 30min., or 4ºC (on ice) overnight, then centrifuge at max. speed
for 30 min at 4ºC.
11.
Remove supernatant and wash the RNA pellet with 1 ml of ice-cold 75% EtOH (made
with RNase-free water).
12.
Centrifuge at max. speed for 10 min at 4ºC.
13.
Carefully remove supernatant and briefly air-dry pellet (5-10min).
14.
Resuspend in 25-50l of DEPC-treated water (RNase-free).
15.
Quantitate RNA by spectrophotometer (A260)
16.
Calculate the amount of RNAse-free DNAse to add as follows:
Add enough DNAse to digest all of the quantitated RNA (ensures complete gDNA
digestion.)
For example: if you calculated that your sample contains 50ug of RNA,
then you need to add 50 units of DNAse to the tube. The DNAse from
Ambion is supplied at 2U/ul, so you would add 50÷2 = 25ul of DNAse to the prep.
17.
Set up DNase Digestion Reaction:
RNase-free DNase (1/10th volume of total reaction, Ambion)
10x DNase Buffer (Ambion)
RNA sample
DEPC water to total volume
18.
Mix and incubate at 37C for 30min.
19.
Add an equal volume of Trizol to the DNase digestion reaction and 1/10th volume
Chl/IAA, cover tubes tightly and shake vigorously for 15 sec.
20.
Repeat steps #6 through #15.
21.
Aliquot and store RNA at -80C.