Total RNA Isolation From 50–100 Mg Plant Tissue

Total RNA Isolation From 50–100 mg Plant Tissue

Method
Cell Lysis
1.Add 50–100 mg (0.05–0.1 g) frozen ground tissue or fresh tissue to a tissue grinder tube on ice and then add 3 ml cell lysis solution. Grind frozen tissue finely in liquid nitrogen with a porcelain mortar and pestle. Keep tissue frozen until added to the tissue grinder tube.

2 . Homogenize quickly using 5–10 strokes with a tube pestle. Transfer sample to an Oak Ridge centrifuge tube (or other tube rated for high speed).

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

2.Invert tube 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).

2. Cap the tube. Mix the sample by inverting gently 50 times.

3. Centrifuge at 15,000 x g for 5 min; the RNA will be visible as a small, translucent pellet.

4. Pour off supernatant and drain tube on clean absorbent paper. Add 3 ml 70% ethanol. Cap and invert the tube 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 to air-dry for 15 min.

RNA Hydration
1. Add 300 µl RNA hydration solution (300 µl will give a concentration of 500 µg/ml if the total yield is 150 µg RNA).

2. Allow RNA to rehydrate on ice at least 30 min. Vortexvigorously for 5 sec, pulse-spin and carefully transfer sample to a 1.5 ml microfuge tube. Store purified RNA sample at - 7 0 ° C t o -80°C until use.

3. Before use, vortex sample vigorously for 5 sec and pulsespin. Pipet sample up and down several times to ensure adequate mixing.

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