( AquaPure R...)Total RNA Isolation From 500 l Body Fluid

Method
Cell Lysis
1. Vigorously add 2.5 ml cell lysis solution to an Oak Ridge centrifuge tube (or other tube rated for high speed) containing 500 l body fluid (e.g., cerebrospinal fluid, plasma, saliva, serum, sputum, synovial fluid, urine, whole blood).

2. Vortex for 5 sec to mix thoroughly.

3. Heat sample to 65C for 5 min to complete lysis. Cool sample to room temperature. (This step is optional when testing for certain viruses such as hepatitis C.)

Protein-DNA Precipitation
1. Add 1 ml protein-DNA precipitation solution to the lysate.

2. Cap tube and invert gently 10 times and place tube into an ice bath for 10 min. 3. Centrifuge at 15,000 x g for 5 min. The precipitated proteins and DNA will form a tight pellet.

RNA Precipitation
1. Pour the supernatant containing the RNA (leaving behind the precipitated protein-DNA pellet) into a clean Oak Ridge centrifuge tube (or other tube rated for high speed) containing 3 ml 100% isopropanol (2-propanol). If RNA yields are expected to be low, add a glycogen solution as a carrier to the isopropanol (5 l of 20 mg/ml glycogen per 3 ml isopropanol).

2. Mix the sample by capping and inverting gently 50 times.

3. Centrifuge at 15,000 x g for 5 min; depending on yield, the RNA may or may not be visible as a small, translucent pellet.

4. Pour off supernatant and drain tube briefly on clean absorbent paper. Add 3 ml 70% ethanol, cap the tube, and invert it several times to wash the RNA pellet.

5. Centrifuge at 15,000 x g for 2 min. Carefully pour off the ethanol.

6. Invert and drain the tube on clean absorbent paper and allow sample to air-dry for 15 min.

RNA Hydration
1. Add 100 l RNA hydration solution (100 l will give a concentration of 100 g/ml if the total yield is 10 g RNA).

2. Allow RNA to rehydrate for at least 30 min on ice. Alternatively, store purified RNA sample at -70 to -80C until use.

3. Before use, vortex sample vigorously for 5 sec and pulsespin. Pipet sample up and down several times to mix the sample further.

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