There are two different scale of RNA extraction: small scale and big scale. And also there are two methods for RNA extraction: conventional (phenol method) and modern (using kit solution) method. This time I will share about How to Extract RNA using conventional method. We need extra time to finish up all the steps. The steps are as follow:
- Grind 3-5 g of frozen plant tissue in liquid N2 in a mortar using a pestle to a fine powder, then add 5 mL of water-saturated-phenol and 5 mL of extraction buffer.
- Thaw the sample and transfer to Falcon tube using micro-pipette.
- Vortex well and centrifuge 10 min at 3,000 rpm, room temperature.
- Transfer aqueous phase to a new tube, and add 2.5 mL of phenol and 2.5 mL of chloroform, then vortex well.
- Centrifuge at 3,000 rpm for 10 min and transfer aqueous phase to a new tube, add 5 mL chloroform, then vortex well.
- Repeat step 5 until chloroform phase is clean.
- Divide the RNA solution into two new tubes.
- Add 0.1 vol of 3M sodium acetate, pH 5.2 and 2.5 vol of 100% ethanol and place 30 min at -70oC.
- Centrifuge 10 min at 10,000 rpm, 4oC.
- Decant ethanol and add 1 mL of DEPC-water to dissolve RNA.
- Electrophorese to confirm RNA (optional).
- Add 3 mL of DEPC-water and 5 mL of 4M LiCl, and then mix well.
- Freeze 30 min at -70oC and centrifuge 10 min at 10,000 rpm, 4oC.
- Discard the supernatant carefully, and redissolve RNA in 0.4 mL of DEPC-water, then transfer to a 1.5 mL microcentrifuge tube.
- Quantitation of RNA with absorbance: Concentration of RNA (mg/mL) = A260.
Note: Extraction buffer: 1 mL of 1M Tris-HCl, pH 9.0, 1 mL of 2M LiCl, 0.25 mL of 0.5M EDTA, pH 8.2, 0.5 mL of 10% SDS, and 2.25 mL of DEPC-water.