Preparation of RNA from tissues with Trizol (PJ Keller)

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RNA from Tissue with Trizol (Click to Download)

*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’
  19. Dilute a fraction at 1:100 in 10 mM Tris pH 8 and measure concentration

Aliquot small fractions of RNA and store at -80

RNA preparation from plates with Trizol (PJ Keller)

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RNA Prep Trizol (Click to Download)

  1. Aspirate media, wash plates with PBS 1X and aspirate off excess liquid
  2. Add 0.5 ml Trizol to each well in the hood, pipet up and down a few times to cover the wells, pipet the mixture into autoclaved microfuge tubes, incubate for 5’ at RT (combine the replicates into one tube)
  3. Add 0.2 ml Chloroform to each tube, cap tightly and shake vigorously for 15 seconds, incubate at RT for 2-3 min.
  4. Spin at 12,000 rpm for 15 minutes at 4C, RNA will be in the aqueous top phase.
  5. Transfer the aqueous phase to a fresh microfuge tube
  6. Add 500 ml of RNA only isopropanol to each tube, incubate at RT 10’
  7. Spin at 12,000 rpm for 10 minutes at 4C, look for RNA pellet
  8. Wash the pellet once with 1ml 75% ethanol prepared with Rnase-free water, vortex the sample to mix and spin at 7500 rpm for 5 min
  9. Remove the ethanol, invert tube and allow to air dry for 10-15 min
  10. Resuspend the pellet in 50 ml of Rnase-free water, incubate for 10’ at 50C to dissolve, store at –80