Molecular Techniques and Methods

Isolation of Total RNA from Paraffin-Embedded Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In fixed and paraffin-embedded tissues which are rich in RNases, RNA fragments of 100 bases in length are still present.

In this procedure, paraffin-embedded tissues are deparaffinized and hydrated. Any enzymes including RNases and proteins in tissue sections are digested with a protenase K. Nucleic acids is extracted by acid phenol: chloroform: IAA and precipitated in alcohol.




MATERIALS AND SOLUTIONS

Proteinase K Buffer (100 ml)
1.6 M Guanidine thiocyanate ---------------------------- 18.9 g
30 mM Tris-HCl (pH 7.6) ------------------------------ 3 ml of 1 M Tris-HCl
0.72% Sodium N-lauryl sarcosine ----------------------- 7.2 ml of 10 % Sarkosyl
Add DEPC-treated H2O to make a final volume of ---- 100 ml


Digestion Buffer (10 ml)
2-Mercaptoethanol ---------------------------------- 30 ul
Proteinase K buffer ---------------------------------- 10 ml


Proteinase K Solution (20 mg/ml)
Proteinase K ---------------------------------------- 20 mg
50% Glycerol --------------------------------------- 1 ml
  • Store at 20oC


    Acid phenol: chloroform: IAA (70:30:1)




    PROCEDURES

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

    1. Cut the paraffin sections into 10-15 um thick with microtomes.

    2. Place the 3-5 cut sections in 1.5 ml microfuge tubes.

    3. Solubilize the paraffin with 1 ml of xylene for 5 minutes at room temperature.

    4. Repeat step 3.

    5. Centrifuge for 10 minutes at 9,000g. Save the tissue pellet.

    6. Wash the tissue with 1 ml of absolute ethanol for 10 minutes.

    7. Wash the tissue with 1 ml of 95% ethanol for 10 minutes.

    8. Centrifuge for 10 minutes at 9,000g. Save the tissue pellet.

    9. Discard the ethanol and air-dry the tissue.

    10. Add to each air dried tissue 1 volume (100-300 ul) of Digestion Buffer.

    11. Add proteinase K to a final concentration of 6 mg/ ml.

    12. Incubate overnight at 45oC.

    13. Add 1 volume of Acid phenol: chloroform: IAA (70:30:1).

    14. Vortex and put in ice for 15 minutes.

    15. Centrifuge at 13,000g for 20 minutes.

    16. Save the upper aqueous phase.

    17. Add 5 ul of Glycogen (10 ug/ul) and 1 volume of isopropanol to the aqueous phase.

    18. Precipitate RNA at -20oC for 48-72 hours.

    19. Centrifuge at 13,000g for 30 minutes at 4oC.

    20. Wash the pellet in 100 ul of 75% ethanol.

    21. Centrifuge at 13,000g for 5 minutes.

    22. Air-dry the pellet.

    23. Resuspend the RNA pellet in 10-30 ul of DEPC-treated H2O.

    24. Store at -80oC.




    NOTES

  • To handle the paraffin sections more easily, it is better to obtain rolled up sections when they are cut. This may be obtained by decreasing the temperature of the paraffin blocks putting them in a freezer before cutting.


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





  • KIT INFORMATION




    REFERENCES

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

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