RNA Isolation Protocols -- also see mRNA Isolation Protocols -- RNA Isolation Troubleshooting
(for cultured
adhesive cells, in 100 mm dish)
1.
2. Add 1 ml trizol, and then scrub the dish bottom.
3.
Transferred the lysate to 1.5 ml eppendorf tube, and keep on ice.
4. Add 0.2 ml chloroform, and vertex for 30 seconds.
5. Incubate on
ice for 5-10 minutes.
6. Centrifuge at 14000 rpm for 15 minutes at 4 centigrade.
7. Transfer aqueous phase to new eppendorf
8. Add
0.5 ml isopropanol.
9. Incubate on ice for 10 minutes.
10.Centrifuge at 14000 rpm for 15 minutes at 4 centigrade.
11.Discard the supernatant.
The pricipitated pellet is raw RNA.
12.Wash the RNA pellet with 0.5 ml 75% ethanol. Centrifuge at
9000 rpm for 5
minutes at 4 centigrade. Discard the supernatant.
13.Repeat step 13 one more.
14.Air dry the pellet for about 10 minutes. (Note: don't
overdry,
otherwise, it will be very difficult to dissolve it.)
15.Rehydrate with 20-50 micro liter DEPC water.
Spectrophotometric Determination of RNA Concentration
RNA concentration (ug/ml)=OD260nm x 40 x dilution
Purification: If the isolated RNA is pure enough, the ratio of
OD260nm/OD280nm should be between 1.8 to 2.0
The RNA can be purified futher by using a specific column, or DNAse treatment.
You also can confirm the RNA quality by running
a mini-gel.
This method can be used for bigger or smaller dishes. You can adjust the
reagent volume accordingly.
This method
can also be used for tissue. The tissue should be homogenized before
adding trizol.
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