The following protocol is a modified version of the Trizol protocol for RNA isolation from plant tissues in which a high-salt isopropyl fermentation precipitation step is added to selectively precipitate the RNA while leaving the polysaccharides and proteoglycans in solution. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Preparation of RNA from plant tissues using Trizol
Materials and Instruments
Plant tissue Move I. Materials For more product details, please visit Aladdin Scientific website.
Trizol Chloroform Isopropyl alcohol Ethanol DEPC-treated water Liquid N2 NaCl Sodium citrate
Rotor-stator homogenizer Mortar and pestle Conical tube
1. Buffers, solutions and reagents
Trizol (Invitrogen)
Chloroform
Isopropyl alcohol
Ethanol, 70%, prepared with diethyl pyrocarbonate (DEPC)-treated water
DEPC-treated water,
Liquid N DEPC-treated water, liquid N
NaCl,1.2mol/L
Sodium citrate, 0.8mol/L
2. Special equipment
Rotor-stator homogenizer (or equivalent)
Mortar and pestle, washed in DEPC-treated water, pre-frozen in liquid nitrogen
50 ml conical tubes, e.g. Falcon
3. Cells and tissues
Suitable plant tissues
II. Methods
1. Grind approximately 1g of plant tissue in a liquid nitrogen pre-frozen mortar and pestle, using enough liquid nitrogen to submerge the plant tissue during the operation.
It is important to grind the tissue to a very fine powder.
2. Transfer the powder to a 50 ml conical tube to which Trizol has been added at 10 ml per gram of tissue.
3. Homogenize the tissue for 30 s using a rotor-stator homogenizer.
4. Centrifuge the lysate at 1500 g at 4°C for 15 min to remove the residue.
5. Transfer the supernatant to a new tube. Add 200ul of chloroform per ml of Trizol.
6. Invert the tube several times to mix. Allow to incubate at room temperature for 5 min.
7. Centrifuge at 1500 g for 20 min at 4°C. Transfer the supernatant to a new 50 ml tube.
8. Add 0.5 times the volume of isopropyl yeast and 0.5 times the volume of 0.8 mol/L sodium citrate, 1.2 mol/L sodium chloride solvent.
For example, for a 500ul volume of liquid phase, add 250ul of isopropyl alcohol and 250ul of sodium citrate solution.
9 Mix and incubate at room temperature for 1 min. Storage at -20°C overnight increases RNA yield.
10. Centrifuge at 1500 g at 4°C for 20 min.
11. Remove the supernatant and add 1 ml of 70% ethanol per ml of Trizol for initial extraction.
12. Centrifuge at 1500 g for 15 min. Dispose of the supernatant carefully to avoid disturbing the sediment. Air-dry the sediment.
13. Dissolve the RNA in DEPC-treated water.
The RNA yield of lg of mature leaf tissue can be up to 500ug.
This protocol may not work for plant tissues containing polyphenolic compounds or resins. ConcertPlantReagent (Invitrogen) is recommended for plant tissues rich in polyphenols, resins, or starch that are difficult to extract.