Molecular Techniques and Methods

Isolation of Total RNA from Animal Cells and Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this procedure, RNA can be isolated from large amounts of material or can easily be miniaturized to a RNA minipreparation for small amounts of material. High salt (3 M LiCl) and urea (6 M) effectively inhibit ribonulceases from materials. Lithium chloride also selectively precipitates RNA, while other components (DNA, polysaccharides, and proteins) remain in solution.


MATERIALS AND SOLUTIONS

LiCl/ Urea Solution (100 ml)
3 M LiCl ------------------------------------------------ 30 ml of 10 M LiCl
6 M Urea ------------------------------------------------ 36 g
Add DEPC-treated H2O to make a final volume of ----- 100 ml


Acid phenol: Chloroform: IAA (25: 24: 1)


3 M Sodium acetate (pH 4.8)
Adjust pH 4.8 with diluted acetic acid


Phosphate-Buffered Saline Solution (PBS)
Store at 4oC.




PROCEDURES

  • All manipulations should be performed on ice! Always wear gloves!

    1. Wash cells or tissues with 5 ml ice-cold PBS two times.

    2. Add 5 ml ice-cold LiCl/ Urea solution to the cells or tissues.

    3. Transfer cells or tissues to a Dounce homogenizer and homogenize the probes by 15-20 strokes in the homogenizer.

    4. Transfer the samples to 16 ml polypropylene tubes and let stand at 4oC overnight.

    5. Centrifuge the samples at 13,000g for 30 minutes at 4oC.

    6. Discard supernatants.

    7. Add 2.5 ml LiCl/ Urea solution and vortex thoroughly to dissolve the RNA pellet.

    8. Centrifuge the samples at 18,000g for 30 minutes at 4oC.

    9. Discard the supernatant and add 2.5 ml of STE buffer.

    10. Vortex to dissolve the RNA pellet.

    11. Add 2.5 ml Acid phenol: Chloroform: IAA (25: 24: 1) and vortex thoroughly.
  • The solution will become turbid due to the water-insoluble organic solvents and the SDS precipitation.

    12. Centrifuge the samples at 13,000g for 20 minutes at 4oC.

    13. Transfer the supernatant to a new tube.

    14. Add 0.1 vol of 3 M Sodium acetate (pH 4.8) and mix well.

    15. Add 2.5 vol of absolute ethanol and keep at -70oC for 30 minutes.

    16. Centrifuge the samples at 18,000g for 30 minutes at 4oC.

    17. Discard the supernatant.

    18. Wash the RNA pellets with 5 ml 70% ethanol.

    19. Centrifuge the samples at 18,000g for 10 minutes at 4oC.

    20. Air dry RNA pellet.

    21. Dissolve the RNA in 100-500 ul DEPC-treated H2O.

    22. Isolate poly A(+)-RNA, if it is necessary.




    NOTES

  • Do not over-dry the RNA pellet. It will be very difficult to dissolve the RNA pellet.





  • KIT INFORMATION




    REFERENCES

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

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified