A technique for extracting samples of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) for use in diagnosing and studying infectious and genetic diseases has been developed. The technique enables the concentration and purification of nucleic acids from large (in comparison with older techniques) volumes of bodily fluids or digested tissues, with minimal nuclease activity and minimal loss of the nucleic acids.
The technique involves the use of a centrifuge equipped to handle 15-milliliter polypropylene conical tubes that are standard equipment items in biomedical research. Fresh or frozen samples with volumes up to 4 milliliters can be used without prior concentration steps and without numerous microfuge tubes; these features minimize the loss of nucleic acids and the cross-contamination of samples, both of which are observed when numerous concentration steps and numerous tubes are used. Isolation of DNA and/or RNA can be accomplished in as little time as 40 minutes.
The technique involves the use of an extraction solution of the following composition:
- 1 part by volume of a solution that has a pH of 7.0 and that contains (a) guanidinium thiocyanate at a concentration 4 M, (b) sodium citrate at a concentration of 25 mM, (c) sarcosyl at a concentration of 0.5 percent by volume, and (d) 2-mercaptoethanol at a concentration of 0.1 M;
- 0.1 part by volume of a 0.2-M solution of sodium acetate;
- 1 part by volume of an aqueous solution of phenol at a pH of 7.9; and
- 0.2 part by volume of chloroform.
The following are the steps of the extraction procedure according to this technique:
- Add the extraction solution to each sample.
- Transfer the entire volume sample (usually 2 to 3 mL) of each sample to one of the 15-mL polypropylene conical tubes.
- To each such tube, add 2.5 mL of RNAzol™ and 0.25 mL of R-chloroform, then vortex for 20 seconds.
- Put each such tube on ice for ten minutes.
- Spin the tubes in the centrifuge at a speed that yields a centripetal acceleration of 10,000 times normal Earth gravitation for 10 minutes.
- Take the top layer of each sample that has been centrifuged, add an equal volume of isopropyl alcohol, and vortex briefly.
- Let each such sample stand for at least 2 hours at a temperature of –20 °C to allow precipitation to take place. Alternatively, faster precipitation of nucleic acids can be achieved through addition of ammonium acetate.
- Perform centrifugation (again at 10,000 times normal Earth gravitation) for 10 minutes on the samples that have undergone the precipitation treatment.
- Remove and discard the liquid from each sample, which now takes on the form of a pellet.
- Wash each pellet by gently overlaying it with an aqueous solution of 75 percent ethanol.
- Decant the liquid.
- Use a disposable laboratory towel wrapped around forceps to wipe, from inside each sample tube, the solid sample material left behind by decanting of the liquid.
- Resuspend the solid material in 30 µL of a buffer solution of 1x tris-ethylenediaminetetraacetic acid (EDTA).
This work was done by Duane L. Pierson ofJohnson Space Center and Raymond P. Stowe. For further information, access the Technical Support Package (TSP) free on-line at www.nasatech.com/tsp under the Bio-Medical category.