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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.4/2.1

TITLE: THE ISOLATION OF RNA FROM CULTURED CELL LINES FOR SUBSEQUENT USE IN RT-PCR ASSAYS

PROCEDURE

    Cell Preparation

  1. a Suspension cultures

    2. Collect 1x107 cells approximately in a sterile centrifuge tube and spin at 200g for 5 min.

    b Monolayer cultures

  2. Pour off the culture media from a 75cm2 tissue culture flask containing a confluent monolayer. Wash twice with a 1x PBS. Add 0.8ml pre-chilled denaturing solution to the flask. Remove the resulting lysate to microcentrifuge tubes.

    3. RNA Extraction

  3. Add 0.60ml 2M sodium acetate pH 4.0 to each tube and mix thoroughly by inversion.
  4. Add 600ml phenol:chloroform:isoamyl alcohol (25:24:01) to each tube. Mix thoroughly by inversion and shake vigorously for 10 secs. Chill on ice for 15 min.
  5. Centrifuge at 13000g for 10 min.
  6. Remove the top aqueous layer containing the RNA and transfer it to another microcentrifuge tube. (DNA and proteins will remain in the organic layer at the interface).

    7. Precipitation

  7. Add an equal volume of isopropanol and incubate the sample at -20°C for 1 hr at least. For low levels of RNA incubation overnight is recommended.
  8. Pellet the RNA at 13000g for 10 min, pour off the isopropanol and pool the pellets.
  9. Resuspend both pellets in 0.5ml denaturing solution and vortex until the RNA is dissolved. In some cases brief heating to 65°C may be required.
  10. Add an equal volume of isopropanol and recipitate as before.
  11. Pellet the RNA by centrifugation at 13000g for 10 min.
  12. Wash the pellet with 1ml ice cold 75% ethanol and centrifuge as above.
  13. Dry the pellet for 10-15 min in a 37°C incubator. Do not let the pellet dry out completely, but ensure all ethanol has evaporated.
  14. Resuspend the pellet in 100ml to 200ml RNase free distilled water and store at 20°C.

Denaturing Solution: CBS Buffer:

  • 25g Guanidine isothiocyanate 42mM sodium citrate
  • 33ml CBS buffer 0.8% n-lauryl sarcosine
  • 0.2mM B-mercaptoethanol

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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