Preparation of Total RNA Based on

1. There are a number of quite good procedures for preparing high-quality RNA. In addition, several commercial kits are available for this purpose that work reasonably well, but are rather expensive. The method appropriate for your cells or tissue depends largely on the amount of endogenous RNases present. The following method is somewhat tedious but always gives high-quality RNA.

2. Homogenization—the tissue is homogenized in 5 vol of ice-cold GuSCN solution on ice with two 30-s bursts of the polytron at maximum speed. Be sure that the tissue is completely dispersed. The size of polytron generator used depends on the quantity of tissue, but for most cases, the 1-cm type is adequate. For hard tissues, such as bone, a generator with blades should be used. Cultured cells are first trypsinized, rinsed with PBS, and resuspended in a minimum volume of PBS. Each 1 mL of densely suspended cells is homogenized in ~10 mL of ice-cold GuSCN solution on ice as above. It is convenient to use sterile, disposable 50-mL polypropylene tubes for both homogenization and extractions.

3. Extract the homogenate with phenol:chloroform once and centrifuge to separate the layers for 5 min at 3000g. The upper, aqueous layer will appear somewhat milky, and is removed to a fresh tube. If you just use phenol instead of phenol chloroform, there will be no phase separation.

4. Extract the aqueous layer once with an equal volume of chloroform:isoamyl alcohol and centrifuge as above.

5. Centrifugation—the homogenate is layered onto 2.2-mL cushions of CsCl-EDTA solution prepared in SW-41 tubes. Centrifugation is for 18 h at 28,000 rpm at 20°C in an SW-41 rotor.

Fig. 1. Schematic view of the steps in constructing a subtracted cDNA library or subtracted probe. (See color plate 1 appearing after p. 368.)

If an SW-28 or SW-27 rotor is used, then the centrifugation speed should be decreased to 22,000 rpm. If other rotors are used, the volume of the cushion should be one-fifth of the total tube volume. Be sure to check the rotor manual to determine the maximum rotor speed with concentrated CsCl solutions.

6. Collection of the RNA—the tubes are removed from the centrifuge and placed in a rack.

7. The GuSCN layer is aspirated down to and including ~0.5 mL of the cushion. The tube is carefully filled with DEPC containing H2O, allowed to stand 2-5 min, and then the water is aspirated. This is repeated twice.

8. After the final rinse, the tube is quickly inverted and allowed to drain. The bottom 2 cm of the tube are cut off with a fresh scalpel, and the tube placed upright in a rack. The RNA appears as a glistening button in the center of the tube. Small amounts of RNA may not be visible. The pellet is carefully rinsed with 0.5 mL of DEPC-treated H2O, and the tube inverted to dry.

9. The pellet is then macerated with, and taken up into a tip containing ~100 |L of DEPC-treated H2O (or an appropriate amount for the RNA yield you end up with). The RNA is pipeted up and down and transferred to an Eppendorf tube. Be sure you transfer all the RNA. The RNA is then heated at 70°C for 2-30 min until dissolved. Large amounts of RNA need longer times and require more H2O. The insoluble debris is removed by brief centrifugation, and the supernatant removed to a fresh Eppendorf tube.

10. Add 0.5 vol of NH4-acetate and 2 vol (i.e., 2X the volume of the RNA + acetate solution) of absolute ethanol. Precipitate for 15 min at -70°C or several hours to overnight at -20°C (preferred method). Centrifuge for 15-30 min at 4°C, and then drain the supernatant. Rinse the pellet three times with the ethanol-sodium acetate solution, centrifuging briefly between rinses.

11. Rinse the pellet once with 80% ethanol, once with 100% ethanol, and air-dry. Resuspend the RNA in an appropriate volume of H2O, and store at -70°C.

Was this article helpful?

0 0

Post a comment