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 l RNase-free H20, remove 10 l 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 l BME to 3.5 ml RLT
11. Add 350 l RLT to 100 l Trizol RNA sample, mix
12. Add 250 l 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 l RPE, spin 15s
16. Discard the flow through, wash again with 500 l RPE, spin 2’
17. Discard Flow through, spin again 1’
18. Elute into a 1.5 ml collection tube with 50 l 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