Molecular Techniques and Methods

Isolation of Total RNA from Plant Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

Plant tissue has a large amount of polysaccharides and polyphenols, which form complexes with nucleic acids during tissue extraction and coprecipitate during subsequent alcohol precipitation. Depending on polysaccharides and polyphenols, the resulting alcohol precipitates can be gelatinous and difficult to dissolve. An RNA solution contaminated with polysaccharides and polyphenols is viscous and absorbs strongly at 210-230 nm. The contaminated RNA is not suitable for northern analysis, in vitro translation, or reverse transcriptase reaction.

In this procedure, guanidinium thiocyanate is used to lyse cells and to denature proteins including ribonucleases. Most polysaccharides that remain in the aqueous phase are selectively precipitated by potassium acetate. RNA is further purified from residual contaminants by lithium chloride precipitation.

  • The yield of total RNA is 2 mg/g of fresh tissue.



    MATERIALS AND SOLUTIONS

    0.75 M Sodium Citrate Buffer (100 ml)
    0.75 M Sodium citrate ---------------------------- 22 g
    Adjust pH to 5.2 with Citric acid.
    Add distilled H2O to make a final volume of ----- 100 ml
  • Treat 0.1% DEPC (diethyl pyrocarbonate) and autoclave.


    Guanidine Thiocyanate Buffer
    Guanidine thiocyanate ----------------------------- 250 g
    Deionized H2O ------------------------------------ 293 ml
    0.75 M Sodium Citrate Buffer --------------------- 17.6 ml
    10% (w/v) Sarkosyl (N-laurosylsarcosine) --------- 26.4 ml
  • Heat to 65oC to dissolve.


    Extraction Buffer
    Guanidine Thiocyanate Buffer ---------------------- 10 ml
    Polyvinylpolypyrrolidone ---------------------------- 2 g
    2-mercaptoethanol
    (add just before use) ---------- 72 ul


    Acid Phenol (pH 4.5)


    2 M Potassium Acetate (pH 4.8) (100 ml)
    Potassium acetate ----------------------------------- 19.7 g
    Adjust pH to 4.8 by glacial acetic acid.
    Add deionized H2O to make a final volume of ------- 100 ml


    2 M Sodium Acetate (pH4.0)


    10 M LiCl




    PROCEDURES

    1. In liquid nitrogen, grind 2 g of plant tissue in a mortar and pestle to a fine powder.
  • Do not allow the tissue to thaw.

    2. Immediately, transfer a powder to a 100 ml beaker containing 20 ml of Extraction Buffer. Mix well.

    3. Transfer the homogenate into a 50 ml Oak Ridge tube.

    4. Centrifuge at 15,000g for 30 minutes at 4oC.

    5. Transfer the supernatant to 50 ml Oak Ridge tube.
  • Careful - do not disturb the whitish gel-like pellet since it is very soft. Insoluble polysaccharides form a whitish gel-like layer on top of the tissue debris, which forms a dark green pellet.

    6. Add 2 ml of 2 M Sodium acetate (pH 4.0) and mix by vortexing.
  • Add 20 ml of acid phenol (pH 4.5) and vortex for 1 minute.
  • Add 4 ml of chloroform: isoamyl alcohol (49:1, v/v) and vortex for 1 minute.

    7. Incubate the tube on ice for 20 minutes.

    8. Centrifuge at 15,000g for 20 min at 4oC.

    9. Transfer the supernatant to a 50 ml Oak Ridge tube.
  • Add an equal volume of 2 M Potassium acetate (pH 4.8). Mix by vortexing.

    10. Incubate the tube on ice for 1 hour to precipitate polysaccharides.

    11. Centrifuge at 45,000g for 30 minutes at 4oC.

    12. Transfer the supernatant to a 50 ml Oak Ridge tube.

    13. Add 1 volume of absolute isopropanol.
  • Mix and incubate the tube at -20oC for 1 hour.

    14. Centrifuge at 3,000g for 20 minutes at 4oC.

    15. Decant the supernatant.
  • Wash the pellet with 1 ml of 70% ethanol .
  • Centrifuge at 3,000g for 3 minutes at 4oC.
  • Pour off the supernatant as much as the ethanol as possible.

    16. Dissolve the pellet in 1 ml of DEPC-treated water and transfer to a 1.5 ml microfuge tube.

    17. Add 250 ul of 10 M LiCl, and incubate at 4oC for at least 2 hours.

    18. Centrifuge at 10,000g for 20 minutes at 4oC.

    19. Wash the pellet twice in 1 ml of 70% ethanol.

    20. Resuspend the pellet in 400 ul of STE buffer.

    21. Add 1 ml of absolute ethanol. Incubate at -20oC for at least 15 minutes.

    22. Centrifuge at 12,000g for 20 minutes at 4oC.

    23. Wash the pellet twice in 1 ml of 70% ethanol.

    24. Resuspend the pellet in 500 ul of DEPC-treated H2O.

    25. Check the RNA quality.




    NOTES

  • At pH 7.0 or higher DNA and RNA partition into the aqueous phase. At an acidic pH (4.5), DNA is denatured and will move into the organic phase leaving the RNA alone in the aqueous phase.


  • Resolubilizing the RNA pellet after precipitation can be a time-consuming and difficult task. To simplify this task, spin down the pellet and aspirate off the alcohol. Then spin again for 5 seconds to bring down remaining alcohol clinging to the tube walls and aspirate. It is important not to over-dry the sample. Air dry the RNA for 5 to 10 minutes.
    Caution - Do not dry an RNA pellet by vacuum centrifugation in a speed vac.


  • The RNA pellet should be white. The presence of off-white, gel-like pellet indicates contamination by polysaccharides. In such a case, resuspend the pellet in 200 ul of DPEC-treated H2O, add 1 vol of 2 M Potassium acetate (pH 4.8), and incubate on ice for 30 minutes. Centrifuge the microfuge tube at 12,000g at 4oC for 20 minutes. Transfer the supernatant to a new tube, and precipitate the RNA with 0.1 volume of 2 M Sodium acetate (pH 4.0) and 2.5 vol of 100% ethanol at -20oC for 30 min.




  • KIT INFORMATION




    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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