ISOLATION OF RNA FROM TISSUE CULTURE CELLS

The following text describes modifications of a protocol by P. Chomczynski & N. Sacchi entitled "Single-step method of RNA isolation by acid gaunidinium thiocyanate-phenol-chloroform extraction". Anal. Biochem. 162:156-159, 1987. This method is relatively fast and may be used for isolation of RNA from tissues as well as from cells. We typically start with at least 5 x 107 cells that were fed with fresh media the day before harvesting. There are large differences in the amount of RNA in different types of cells with peripheral blo od lymphocytes being among the lowest. All glassware is pretreated by baking at 250°C for 4 hours before use to remove RNAses. All aqueous solutions are pretreated with DEPC except for tris solutions which are made with DEPC-treated H2O.

  1. Pellet cells by centrifugation in a disposable, conical tube at 150-200xg (1000-1200 rpm in table top centrifuges) at room temperature for 6 minutes. Wash the cells once in PBS. For about 5x107 cells, resuspend in 1 ml of solution D. (This volume may be increased if more cells are present or if there is difficulty in getting the cellular material into suspension. For fewer cells, use as little as 0.25 ml of solution D and proportionately reduced volumes of other reagents). Homogenize the cells using a 3 or 5 ml syringe with at least a 1 inch 22g needle. Avoid foaming! For less than 1.5 ml volumes, transfer to a 1.5 ml microcentrifuge tube. For larger volumes, transfer to a Corex tube or a 15 ml Falcon polypropylene tube wrapped in about a 6" strip of parafilm to provide support during centrifugation. For tissue samples, the original protocol suggests mincing freshly excised tissue on ice and homogenizing them at room temperature with 1 ml of solution D in a glass-Teflon homogenizer, followed by transfer to a 4 ml polypropylene tube.
  2. For 1 ml of buffer D, add 100 µl of 2M sodium acetate, pH 4, 1 ml of H2O-saturated phenol, 200 µl of a 49:1 mixture of CHCL3:isoamyl alcohol. Maintain these ratios when varying the volumes of buffer D. Mix thoroughly by inversion after addition of each reagent. Vortex the final mixture vigorously for 10 seconds and transfer to ice for 15 minutes.
  3. Centrifuge 10,000 xg for 20 minutes at 4°C. Transfer the RNA-containing aqueous phase to a fresh tube, and discard the DNA and proteins which should be present in the phenol and at the interface. Avoid pipeting any of the white cloudy interface. It will contain DNA.
  4. Add 1 volume of isopropanol (or two volumes absolute ethanol) to the aqueous phase and precipitate at -20°C for at least 1 hour.
  5. Centrifuge at 10,000 xg for 20 minutes at 4°C. Transfer the pellet with a pipet tip or a sterile Pasteur pipet to a 1.5 ml microcentrifuge tube. Resuspend the pellet in 0.3 ml of solution D. Heating to 65 - 70°C and vortexing will help r esuspend the pellet. Precipitate the RNA again in 1 volume of isopropanol (or two volumes of ethanol) at -20°C for at least 1 hour. Centrifuge at high speed (13,000 xg in a microcentrifuge) for 10 minutes at 4°C. Wash the RNA pellet in 75% ethanol, centrifuge again and vacuum dessicate. Avoid over drying. Resuspend the pellet in DEPC treated TE or H2O. Measure the OD in a spectrophotometer. Life Technologies reports the A280 absorbance of RNA is low w hen it is resuspended in TE versus water (see D. Fox. "Measuring Absorbance of RNA Samples" in Focus 20:37, 1998).
Typical yields from about 5x107 cells are 500-1000µg of RNA with a A260:A280 ratio of 1.8 - 2.2. Store the RNA as a sodium acetate/ethanol precipitate at -70°C.


RECIPES:

Solution D:
Prepare in fume hood.
4 M guanidinium thiocyanate
25 mM sodium citrate, pH 7.0
pH with citric acid? (We used the solution from a US Biochemicals kit)
0.5% sarcosyl
0.1 M 2-mercaptoethanol
Store this solution at room temperature for up to 3 months without the 2-mercaptoethanol, or 1 month with the 2-mercaptoethanol.
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Copyright 1993, 1996, 1997, and 1998 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Isolation of RNA from Tissue Culture Cells. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/rnaisol.html). Oklahoma City. Revision Date: July 28, 1998."