mRNA Extraction from fruit

Modified by Rob Alba on 04/27/04

This RNA extraction method is very effective for isolation of pure mRNA from tissues containing a high amount of polysaccharides and/or phenolic compounds. I have modified the protocol of Chang et al (1993) for isolating large quantities of pure mRNA from tomato and pepper pericarp tissues. The mRNA obtained can be used for subsequent cDNA synthesis, aminoallyl labelling, and hybridization to cDNA microarrays. Citation for original protocol: Chang S, Puryear J, Cairney J (1993) A Simple and Efficient Method for Isolating RNA from Pine Trees. Plant Mol Biol Rep 11: 113-116.

A. Solutions Required (Use DEPC-treated water, baked glassware, and RNAse-free stock chemicals for the preparation of all solutions; use RNAse-free plastic ware and disposable for all protocol steps)

  1. RNA EB (mix well and autoclave): 2% CTAB, 2% PVP (polyvinylpyrrolidone, K ≈ 30), 100mM Tris-HCl (pH 8.0), 25mM EDTA, 2M NaCl, 0.5g/L spermidine
  2. beta-Mercaptoethanol, RNAse-free
  3. Chloroform:Iso-amyl alcohol (24:1), RNAse-free
  4. 10M LiCl, RNAse-free
  5. 10mM Tris-Cl (pH 7.5), RNAse-free
  6. 70% EtoH, ice-cold
B. Protocol
  1. Heat 17mL RNA EB in oakridge tube to 65 oC; add 340uL of beta-mercaptoethanol immediately prior to step 2.
  2. Quickly add 3.5g frozen ground tissue and mix vigorously. CAUTION: Vent tube!
  3. Add 15mL of chloroform:Iso-amyl alcohol and mix vigorously. Vent tube.
  4. Separate phases via 10min @ 13,000rpm (RT). Carefully transfer aqueous phase to new Oakridge tube.
  5. Add 15mL of chloroform:Iso-amyl alcohol and mix vigorously.
  6. Separate phases via 10min @ 13,000rpm (RT). Carefully transfer aqueous phase to new Oakridge tube.
  7. Add 15mL of chloroform:Iso-amyl alcohol and mix vigorously.
  8. Separate phases via 10min @ 13,000rpm (RT). Carefully transfer aqueous phase to new Oakridge tube.
  9. Add 15mL of chloroform:Iso-amyl alcohol and mix vigorously.
  10. Separate phases via 10min @ 17,000rpm (RT). Carefully transfer aqueous phase to new Oakridge tube.
  11. Add 1/4 volume of 10M LiCl to aqueous phase and mix by gentle inversion.
  12. Precipitate the total RNA overnight at 4 oC.
  13. Pellet the total RNA via 20min @ 17,000rpm (4 oC) and carefully dump supernatant.
  14. Wash pellet with 10mL of ice-cold 70% EtoH.
  15. Spin 5min at max speed (4 oC) and carefully dump EtoH.
  16. Re-wash pellet with 10mL of ice-cold 70% EtoH.
  17. Spin 5min at max speed (4 oC) and carefully dump EtoH.
  18. Dry RNA pellet for 5 to 10min on lab bench.
  19. Re-suspend total RNA pellet in 10mM Tris-Cl (pH 7.5) such that the [total RNA] ≈ 0.75ug/ul.
  20. Extract mRNA from 100ul of total RNA using oligo d(T)25 Dynabeads (Dynal Inc., catalogue #610.05) via protocol contained in the Dynabead package/kit. Elute mRNA from beads with 10ul (per 3.5g tissue) of eluting solution during final step of mRNA extraction.
  21. Repeat extraction of mRNA from identical total RNA samples until all 300ul have been extracted; combine all three 10ul extracts into a single mRNA extract.
  22. Conduct spec assay to assess mRNA purity, mRNA concentration, and mRNA yield (ug/g tissue FW)
  23. Inspect mRNA integrity via denaturing gel electrophoresis (I typically run 1ug of mRNA in these gels to assess rRNA contamination and the possibility of RNAse degradation).
  24. Store mRNA at -80 oC.