RNA Extraction with TRIZOL®
Wear gloves when handling trizol, work RNase-free!
1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 4ml of Trizol to cells on plastic of 10cm plate
2.5 ml on 60 mm plate
1 ml on 35mm plate
add 3 ml on 3 D culture ( 35 mm plate)
With a 10ml pipette, pipette up and down for 2-3 min until solution is no longer viscous
4. Transfer onto a 15 ml polypropylene tube
5. Spin down the EHS samples in Sorvall Centrifuge for 10 min at 9000 rpm at 4 °C ( No dissolved protein will be pelleted), and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ® ( i.e. 0.2 ml Chloroform for each 1 ml Trizol ® )
7. Close tubes firmly and shake and vortex vigorously for at least 15 seconds
8. Let sit at room temperature for 10 minutes.
9. Centrifuge for 15 min at 9000rpm at 4 °C
10. Transfer upper, aqueous phase to fresh tube, avoid transfer of ANY interface!!
11. Add 0.5 volume IPA to the clean supernatant (i.e. 0.5 ml IPA for each ml Trizol) and add 0.5 µl Glycogen for each ml Trizol
12. Mix immediately by inverting tubes 5-8 times
13. Incubate at room, temperature for 5-10 min.
14. Centrifuge at 9000 rpm for 10 min, 4 °C
15. Discard supernatant
16. Washing with 1ml of 75% EtOH (RNase –free) to each tube
17. Resuspend dry pellet in nuclease-free water and keep on ice
EHS samples in 20-30 µl water each
Plastic samples in 60-80
Quantification of RNA
[RNA] in μg/μl = 40X dilution factor XOD