Extraction of RNA from Tomato (Modified from: Griffiths et al 1999, J Exp Bot 50:793)

By Rob Alba on 12/06/05

SOLUTIONS:

1. DEPC-treated MilliQ^® H₂O
2. 1 M Tris-HCl, pH 8.3
3. 1 M Tris-HCl, pH 8.0
4. 1.4 M NaCl
5. 0.5 M EDTA, pH 8.0
6. Phenol Soln
   • 100 g phenol crystals (melt @ 65^oC)
   • 14 ml m-cresol
   • 0.1 g 8-hyrdoxyquinoline
   • 30 ml DEPC-treated H₂O
7. RNA Extraction Buffer
   • 6% (w/v) 4-aminosalicylic acid
   • 1% (w/v) 1,5-naphthalenedisulphonic acid, disodium salt
   • 50 mM Tris-HCl, pH 8.3
   • 5% (v/v) Phenol Soln
8. Phenol/Chloroform Soln
   • 500 g phenol crystals (melt @ 65^oC)
   • 0.5 g 8-hydroxy-quinoline
   • 500 ml chloroform
   • 20 ml iso-amyl alcohol
     • Prep: Mix components and then equilibrate with 200 ml 100 mM Tris-HCl, pH 8.0. Equilibration: Stir for 1 hr, allow phases to separate, remove upper phase (Tris layer), repeat equilibration twice more, but leave ~100 ml of Tris layer after final equilibration step.
9. 3 M Sodium Acetate, pH 6.0
10. 100% EtOH, 70% EtOH
11. 2X CTAB Extraction Buffer
    • 1.4 M NaCl
    • 2% CTAB (w/v)
    • 100 mM Tris-HCl, pH 8.0
    • 20 mM EDTA, pH 8.0
12. 4X CTAB Precipitation Buffer
    • 1% CTAB (w/v)
    • 50 mM Tris-HCl, pH 8.0
    • 10 mM EDTA, pH 8.0
13. Chloroform
14. RQ1 DNAse (1 µl); 10X RQ1 DNAse Rxn Buffer [400mM Tris-HCl (pH 8), 100 mM MgSO4, 10 mM CaCl2]

1. Add 16 ml of RNA extraction buffer to an Oakridge tube; add 13 ml of phenol/chloroform soln. Add 4 g of frozen "powdered" tomato tissue, cap tube and shake vigorously. Centrifuge at 13,000 rpm (15 min, RT).
2. Transfer the aqueous phase to clean Oakridge tube; add 13 ml of phenol/chloroform soln, cap tube and shake vigorously. Centrifuge at 13,000 rpm (15 min, RT).
3. Transfer the aqueous phase to clean Oakridge tube; add 13 ml of phenol/chloroform soln, cap tube and shake vigorously. Centrifuge at 13,000 rpm (15 min, RT).
4. Transfer aqueous phase to clean Oakridge tube; add 0.1 vol 3M Na-acetate (pH 6.0) and 3 vol ice-cold 100% EtOH. Precipitate for 1 hr at -20^oC.
5. Centrifuge at 13,000 rpm (15 min, 4^oC). Remove supernatant and wash pellet with 5 ml of 70% EtOH. Centrifuge at 16,000 rpm (5 min, 4^oC).
6. Remove all supernatant and allow pellet to dry on lab bench for ~5 min. Re-suspend pellet in 2 ml of DEPC-H₂O and vortex. Heat sample at 50^oC for 10 min and transfer to 14 ml tube.
7. Add 2 ml of 2X CTAB Extraction Buffer. Add 4 ml of 4X CTAB Precipitation Buffer. Invert tube gently to mix and centrifuge at 10,000 rpm (30 min, RT).
8. Remove supernatant and re-suspend pellet in 400 µl of 1.4 M NaCl. Transfer to microfuge tube. Add 1 ml of ice-cold 100% EtOH and precipitate overnight at -20^oC.
9. Spin at max speed in microfuge for 10 min (4^oC). Remove supernatant and wash pellet with 500 µl of 70% EtOH. Spin at max speed for 5 min (4^oC), remove all supernatant, and allow pellet to dry on lab bench for 3 min.
10. Add 500 µl of DEPC-H₂O to pellet and incubate at 50^oC for 10 min; vortex well.
11. Add 500 µl of phenol/chloroform soln. Vortex well and spin at max speed (5 min, RT); transfer aqueous phase to new microfuge tube.
12. Add 500 µl of phenol/chloroform soln. Vortex well and spin at max speed (5 min, RT); transfer aqueous phase to new microfuge tube.
13. Add 300 µl of chloroform, vortex well, and spin at max speed (5 min, RT); transfer aqueous phase to new microfuge tube.
14. Add 0.1 volume of 3M sodium acetate (pH 6.0) and 3 volumes of ice-cold 100% EtOH. Precipitate for 1 hr at -20^oC.
15. Spin at max speed in microfuge for 10 min (4^oC). Remove supernatant and wash pellet with 500 µl of 70% EtOH. Spin at max speed for 5 min (4^oC), remove all supernatant, and allow pellet to dry on lab bench for 3 min.
16. Re-suspend pellet in 179 µl of DEPC-H₂O. Add 20 µl of RQ1 DNAse Reaction Buffer followed by 1 µl of RQ1 DNAse. Incubate for 30 min at 37^oC.
17. Add 300 µl DEPC-H20. Extract with 500 µl of phenol/chloroform soln and spin at max speed in microfuge (5 min, RT). Transfer aqueous phase to new microfuge tube.
18. Extract with 300 µl of chloroform and spin at max speed in microfuge (5 min, RT). Transfer the aqueous phase to new microfuge tube.
19. Add 0.1 volumes of 3M sodium acetate, pH 6, and 3 volumes of ice-cold 100% EtOH. Precipitate for 1 hr at -20^oC.
20. Spin at max speed in microfuge for 10 min (4^oC). Remove supernatant and wash pellet with 500 µl of 70% EtOH. Spin at max speed for 5 min (4^oC), remove all supernatant, and allow pellet to dry on lab bench for 3 min.
21. Add 25-50 µl of DEPC-H₂O to pellet; incubate at 50^oC for 10 min and vortex well.
22. Assay RNA yield and purity using spectrophotometer. Assay RNA integrity via denaturing gel electrophoresis. Store RNA at -80^oC.