RNA extraction
Extraction from adherent cell culture
1. Lyse cells directly in a culture dish by adding 1 ml of TRIZOL® Reagent to
a 3.5 cm diameter dish, and passing the cell lysate several times through
a pipette (2.5ml for the 10cm diameter dish we use)
Extraction from mice lungs
1. Homogenize the lung in 3ml of TRIZOL® Reagent using the electric
homogenizer. Between each sample wash the homogenizer:
1x chloroform → 1x 75% EtOH → 1x H2O DEPC
2. Incubate for 5 min at R.T
3. Add 200μl chloroform per 1ml TRIZOL reagent
4. Shake vigorously by hand for 15sec
5. Incubate 3 min at R.T
6. Centrifuge at 12,000g for 15 min at 4оC
7. Following centrifugation, the mixture separates into a lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase.
RNA remains exclusively in the aqueous phase.
8. Transfer the aqueous phase into a new tube
9. Add 500μl isopropanol per 1ml TRIZOL reagent used
10. Incubate for 10 min at R.T
11. Centrifuge at 12,000g for 10 min at 4оC
12. The RNA precipitate, often invisible before centrifugation, forms a gel-like
pellet on the side and bottom of the tube.
13. Discard supernatant and add 1ml 75% EtOH, mix by vortexing
14. Centrifuge at 7,500g for 5 min at 4оC
15. Discard supernatant
16. Dry the RNA by air
17. Dissolve RNA in DEPC water by passing the solution a few times through
a pipette tip, and incubating for 10 minutes at 55 to 60°C.