P. Keller 12/2007
Preparation of RNA from tissues with Trizol
*Keep tissue on dry ice until homogenization
1. Add trizol to tissue, homogenize with mortar and pestle, pass through a 20G needle 2-3X to shear genomic DNA
2. Incubate for 5 min at RT, keep on ice until all samples are homogenized
3. Add 0.2 ml chloroform per ml Trizol, shake 15 sec, incubate 2-3 min at RT
4. Spin at 12,000 x g for 10’ at 2-8C
5. Transfer the clear aqueous phase to a new tube, add 0.5 ml isopropyl alcohol
6. Incubate at RT 10’, centrifuge at 12,000 x g for 10’ at 2-8C
7. Remove the supernatant, wash the pellet with 1 ml 75% EtOH, vortex
8. Spin down pellet at 7500 x g for 5’ at 2-8C
9. Air dry the RNA pellet 5-10’, resuspend in 110 ml RNase-free H20, remove 10 ml as an aliquot for pre-cleanup analysis
Use Qiagen RNeasy kit to clean up RNA prep (note-may want to add on-column DNAase digestion, see protocol with kit)
10. Make up buffer RLT (3.5 ml) by adding 35 ml BME to 3.5 ml RLT
11. Add 350 ml RLT to 100 ml Trizol RNA sample, mix
12. Add 250 ml 100% EtOH to the diluted RNA, mix
13. Add the sample to a mini column placed in a 2 ml collection tube
14. Spin 15s at 10,000 rpm
15. Transfer the column to a new collection tube, add 500 ml RPE, spin 15s
16. Discard the flow through, wash again with 500 ml RPE, spin 2’
17. Discard Flow through, spin again 1’
18. Elute into a 1.5 ml collection tube with 50 ml RNase-free water, spin 1’
20. Dilute a fraction at 1:100 in 10 mM Tris pH 8 and measure concentration
Aliquot small fractions of RNA and store at -80