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RNA Isolation for Small and Large Scale Samples

Small Scale Isolation

  1. Place up to 20 mg tissue in a 1.5-ml Eppendorf tube.
  2. Add 200 µl denaturing solution.
  3. If there is lots of tissue, use autoclaved glass homogenizer which has been treated with RNaseZap®.
  4. Vortex vigorously until all tissue is broken up and exposed to solution, with no chunks left in solution.
  5. Add 20 µl 2M sodium acetate, pH 4.0, and vortex.
  6. Add 200 µl water-saturated phenol, and vortex.
  7. Add 80 µl chloroform:isoamyl alcohol (49:1), and mix well.
  8. Centrifuge 4°C, maximum speed, 15 minutes.
  9. Transfer aqueous layer to new tube.
  10. Precipitate with 200 µl isopropanol, vortex and let sit on ice for ~10 minutes.
  11. Centrifuge 4°C, maximum speed, 15 minutes.
  12. Pour off supernatant and pipette off extra.
  13. Wash with 75% ethanol, vortex and spin, pour off supernatant and pipette off extra.
  14. Dry, inverted, for 10 minutes at RT.
  15. Resuspend in 20-200 µl 100% formamide. You can also resuspend in DEPC H2O.
  16. Check 1 µl 1:100 or 1:200 by spectrophotometry, being sure to use 1 µl formamide in the blank.

    If you are selecting for poly(A), do it here, then…

  17. DNAse if you are using this for RT-PCR.
  18. Resuspend in 200 µl denaturing solution.
  19. Extract with phenol:chloroform, then with chloroform:isoamyl alcohol.
  20. Precipitate with 200 µl isopropanol.
  21. Wash with 500 µl 75% ethanol, vortexing well.
  22. Centrifuge 4°C, maximum, 5 minutes.
  23. Dry, inverted, 10 minutes at RT.
  24. Resuspend in 20-200 µl 100% formamide.
  25. Check 1 µl 1:100 or 1:200 by spectrophotometry, being sure to use 1 µl formamide in the blank.

Large Scale RNA Isolation

  1. Flash freeze embryos or tissue in liquid nitrogen. Weigh frozen sample.
  2. Grind samples to fine powder using mortar and pestle (which has been treated with RNAseZap or rinsed with 1% SDS and then DEPC H2O), sitting on ice .

    Keep adding liquid nitrogen as you grind, to keep tissue frozen.

  3. Use pre-chilled, cleaned, metal spatula to scrape/scoop powdered sample into 15/50 ml conical with appropriate volume of denaturing solution = 1 mg/10 ml. You can keep adding liquid nitrogen to help collect sample in pestle and scoop out once nitrogen has evaporated. Vortex occasionally to make sure the tissue is fully covered in guanidine isothiocyanate solution as quickly as possible.
  4. Vortex sample to completely expose to denaturing solution.

    Follow steps 5 - 14 above, scaling up volumes as appropriate.

    For centrifugation, use either a glass conical or plastic capped Sorvall tube which has been autoclaved or treated with RNAseZap and washed with DEPC H2O. Spin in Sorvall SA600 at 10K for 15 minutes at 4°C.

  5. Add 20 µl 2M sodium acetate, pH 4.0, and vortex.
  6. Add 200 µl water-saturated phenol, and vortex.
  7. Add 80 µl chloroform:isoamyl alcohol (49:1), and mix well.
  8. Centrifuge 4°C, maximum speed, 15 minutes.
  9. Transfer aqueous layer to new tube.
  10. Precipitate with 200 µl isopropanol, vortex and let sit on ice for ~10 minutes.
  11. Centrifuge 4°C, maximum speed, 15 minutes.
  12. Pour off supernatant and pipette off extra.
  13. Wash with 75% ethanol, vortex and spin, pour off supernatant and pipette off extra.
  14. Dry, inverted, for 10 minutes at RT.

  15. Once pellet has been dried, resuspend in 100% formamide at 4°C in 500-1000 µl. You can also resuspend in DEPC H2O. Let pellet dissolve at RT for 15 minutes or at 4°C O/N.
  16. Check 1:500 sample in H2O by spectrophotometry. Be sure to use 1 µl formamide in the blank.

Denaturing Solution

  • 4M Guanidine Isothiocyanate
  • 25mM Sodium Citrate, pH 7
  • 1M β-Mercaptoethanol (β-ME)
  • 0.5% N-Laurylsarcosine (Sarkosyl)
  • Prepare by dissolving 250 g Guanidine Isothiocyanate in 293 ml H2O, 17.6 ml 0.75M sodium citrate, pH 7, and 26.4 ml 10% Sarkosyl at 65°C with stirring. Keep at RT for up to 3 months.
  • For a working solution, add 0.35 ml β-ME to 50 ml stock solution at time of RNA isolation.

Note: We have found that resuspending the RNA in formamide has increased the longevity of the RNA when stored at 4°C, -20°C and -80°C. RNA resuspended in formamide can be used for northerns, RT-PCR and nuclease protection assays.


© 2014 The University of Texas MD Anderson Cancer Center