Kai Yuet/Protocols:RNA Isolation
RNA Isolation (KPY)
Using Invitrogen's TRIZOL Reagent
For RNA Isolation of Adherent Cells
- 75% Ethanol
- Isopropyl Alcohol
- RNase-free Water
- TE Buffer
- TRIZOL Reagent
- Lyse cells by adding 500 µL of TRIZOL directly to the dish, mixing and passing the cell lysate several times by pipetting.
- Incubate the samples for 5 minutes in Eppendorfs at room temperature.
- Add 100 µL of chloroform per 500 µL of TRIZOL.
- Vigorously shake the microcentrifuge tubes by hand for 15 seconds.
- Incubate the tubes for 2 to 3 minutes at room temperature.
- Centrifuge the tubes at 12000 x g for 15 minutes at 4oC.
- Transfer the aqueous phase (60% TRIZOL by volume) to a fresh Eppendorf.
- Add 250 µL of isopropyl alcohol per 500 µL of TRIZOL to precipitate RNA.
- Incubate the tubes for 10 minutes at room temperature.
- Centrifuge the tubes at 12000 x g for 10 minutes at 4oC.
- Remove the supernatant.
- Resuspend the pellet in 500 µL 75% ethanol per 500 µL TRIZOL and vortex.
- Centrifuge the tubes at 7500 x g for 5 minutes at 4oC.
Redissolving the RNA
- Air dry the RNA pellet and dissolve with TE buffer (200 µL).
- Incubate for up to 10 minutes at up to 60oC to dissolve RNA.
- Store at -80oC.
- Partially dissolved RNA have an A260/280 < 1.6.
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