RNA Preparation

This method is basically that of Chomczynski and Sacchi (2)—it works well from as little as 100-106 cells and it can be scaled up if necessary.

1. Add 100 ||L of solution D to 106 cells or 10 mg tissue in a microcentrifuge tube, and vortex. Most tissues break up almost immediately, but tough tissues may need to be syringed.

3. Add 100 |L of water saturated unbuffered phenol, and mix.

4. Add 20 mL of chloroform, and mix very well. The sample should become cloudy at this stage. If it does not, add extra chloroform.

5. Chill the sample on ice for 15 min, and then spin in a microfuge for 15 min.

6. Take the top phase into a new tube. If one is dealing with a small amount of tissue, one can add 1 |L of RNase-free glycogen at 20 mg/mL to aid precipitation.

7. Add 240 |L of ethanol, and put at -20°C for 30 min.

8. Spin for 15 min in a microcentrifuge.

9 Wash the pellet with 70% ethanol, and drain.

10. Redissolve the RNA in 20 pL H2O and store at -20 or -70°C with the latter being better for long-term storage.

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