RNA Isolation is frequently used in molecular biological experiments. The most important thing for isolate RNA is that you must protect RNA from degradation by RNAase. Here is a brief protocol to isolate RNA from leaf tissue.
Fig. the schematic diagram of RNA isolation principle.
RNA Isolation protocol procedure
- 2 g of frozen tissue is ground to a fine powder with liquid nitrogen using mortar and pestle in presence of 2% insoluble polyvinylpyrollidone (PVPP).
- Powdered sample is transferred to a 50 ml sterile centrifuge tube, to which 20 ml pre-warmed extraction buffer at 65°C is added. Five molar NaCl (0.1 volume) and 1% β-mercaptoethanol to extraction buffer is added just before use.
- The sample tubes are vortexed for 1 min and incubated on ice for 5 min.
- Equal volume of chloroform: isoamylalcohol (24:1) is added, shake for 5 min and centrifuge at 14000 g for 15 min at 4°C.
- The supernatant is transferred to a fresh tube and added 0.1 X of 5 M NaCl, mixed gently, then added 1 X of cold isopropanol and finally precipitated overnight at - 20°C.
- The pellet is obtained by centrifuging at 14000 g for 20 min at 4°C.
- Pellet is washed with 75% ethanol air dried for 10 min and resuspended in 5 ml DEPC treated sterile water.
- Add equal volumes of Phenol:chloroform:isoamylalcohol (25:24:1) to the sample suspension and centrifuged at 14000 g for 15 min at 4°C.
- The supernatant is collected in a fresh centrifuge tube and add 0.1 X of 5 M NaCl, 1 X of cold isopropanol and precipitate overnight at -20°C.
- Pellet is obtained by centrifuging at 14000 g for 20 min at 4°C.
- The pellet is washed with 75% ethanol and resuspended in 500 ul DEPC treated sterile water.
- To the dissolved pellet, equal volumes of chloroform is added shook vigorously for 2 min and centrifuge at 12000 g for 15 min at 4°C and collect the supernatant.
- Add equal volumes of phenol:chloroform (1:1), mix vigorously for 5 min and centrifuge at 12000 g for 15 min at 4°C and collect the supernatant.
- Finally the supernatant is transferred to a fresh 1.5 ml sterile Eppendorf tube and add 0.1 V x 5 M NaCl, 1 X cold isopropanol. Precipitate overnight at -20°C.
- The pellet of RNA is obtained by centrifuging at 12000 g for 20 min at 4°C.
- After washing in 75% ethanol and air dried for 10 min, the pellet is resuspended in sterile water and the RNA sample obtained is stored at -80ºC.