Molecular Techniques and Methods

Isolation of RNA from Cell Suspension Culture

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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

  • In this procedure, the yield of total RNA is 2 ug/mg of cell suspension culture.





    • MATERIALS AND SOLUTIONS

  • Decontaminates all materials and solutions from RNase contamination accoring to the Protocol.



    • SC Buffer (200 ml)
    42 mM Sodium citrate (pH 5.2) ------------- 8.4 ml of 1 M Sodium citrate
    0.83% Sarkosyl ---------------------------- 16.6 ml of 10% Sarkosyl
    DEPC-treated H2O ------------------------ 175 ml


    Denaturing Solution
    4 M Guanidine thiocyanate ------------------ 50 g
    SC Buffer ----------------------------------- 66 ml
  • Filter through 0.2 um millipore membrane and store at 4oC.


    Preparation of Cellulose CF-11 Slurry
    Wash 1 g Whatman cellulose CF-11 with;
  • 20 ml of 0.2 N NaOH,
  • 20 ml of 100 mM Tris-HCl (pH 7.0),
  • and then, 20 ml of DEPC-trated H2O, respectively.
  • Resuspend Whatman cellulose CF-11 in 10 ml DEPC-trated H2O.


    Acid Phenol (pH 4.5): Chloroform: IAA (200: 50: 1, v/v/v)




    PROCEDURES

    1. Collect cultured plant cells in the microfuge tube by centrifugation at 3,000g for 5 minutes.

    2. Add 0.5 ml Denaturing Solution and 5 ul of 2-mercaptoethanol, and grind the cells by pestle.

    3. Add 0.5 ml of Phenol (pH 4.5): CHCl3 : IAA (200: 50: 1, v/v/v).

    4. Vortex for 5 minutes.

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

    6. Transfer supernatent to a new tube.

    7. Add 100 ul Cellulose CF-11 slurry. Mix briefly.

    8. Add 150 ul 3 M Sodium acetate (pH 4.8) and vortex thoroughly for 1 minute.

    9. Centrifuge at 15,000g for 15 min at 4oC.

    10.Transfer supernatant to a new tube.
  • Add 700 ul ice-cold isopropanol and precipitate at -20oC at least 1 hour.
  • Centrifuge at 15,000g for 20 minutes at 4oC.

    11.Resuspend pellet in 500 ul 0.5% SDS and 140 ul 10 M LiCl. Precipitate at 4oC for 3 hours.

    12. Centrifuge at 10,000g for 15 minutes at 4oC.

    13. Resuspend the pellet in 300 ul 0.5% SDS.
  • Use pipet tip to resuspend RNA.

    14. Centrifuge at 15,000g for 5 minutes to remove insoluble materials.

    15. Transfer supernatant to a new tube.
  • Add 30 ul 3 M Sodium acetate (pH 4.8) and 330 ul isopropanol.
  • Precipitate at -20oC for 1 hour.

    16. Centrifuge at 10,000g for 30 minutes at 4oC.
  • Remove residual ethanol by kimwipe.
  • Resuspend in 100 ul 1 mM EDTA.

    17. Check the RNA quality.



    NOTES

  • The purity of RNA can be measured by spectrophotometry.

  • If protein is contaminated in RNA solution, the ratio of absorbances (A260/A280) will be less than 1.9.

  • If polysaccharide is contaminated in RNA solution, the ratio of absorbances (A260/A230) will be less than 2.0.





    • KIT INFORMATION




    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".
    		•
    Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified