Trizol contains guanidine isothiocyanate which can quickly disrupt cells, and at the same time denature the protein in the nucleoprotein complex and release nucleic acid; because the released DNA and RNA have different solubility at a specific pH value and are located in the middle phase and The water phase is used to separate DNA and RNA; after removing the water phase, the pure RNA can be obtained by using organic solvent (chloroform) to extract and precipitate with isopropanol.
1. After the cells are pelleted by centrifugation, the supernatant is discarded, and the cells are lysed to a uniform and bright liquid by repeated pipetting with TRIZOL reagent. The homogenized sample is incubated at 15-30°C for 5 minutes to make the nucleoprotein The body is completely decomposed. (Add 1ml of TRIZOL per 5-10×106 animal cells, plant or yeast cells or 1×107 bacteria. Avoid washing the cells before adding TRIZOL because that will increase the possibility of mRNA degradation.)
2. Separation stage
Add 0.2 ml chloroform per 1 ml TRIZOL. Close the sample tube cap tightly, shake the test tube vigorously by hand for 15 seconds and incubate it at 30°C for 2-3 minutes. Centrifuge at high speed at 2-8°C with a centrifugal force not exceeding 12,000×g for 15 minutes. After centrifugation, the mixture was divided into three layers: the lower red phenol-chloroform layer, the middle layer, and the upper colorless water layer. RNA exists in the water layer without exception. The capacity of the water sample layer is approximately 60% of the added TRIZOL capacity.
3. RNA precipitation
Transfer the water sample layer to a clean test tube, and precipitate the RNA by mixing the water sample layer with isopropanol. When initially homogenized, every 1 ml of TRIZOL corresponds to 0.5 ml of isopropanol. Incubate the mixed samples at 15-30°C for 10 minutes and centrifuge at 2-8°C at a high speed with a centrifugal force of no more than 12,000×g for 10 minutes. RNA precipitation is usually not visible before centrifugation, forming a gel-like sheet-like precipitate attached to the wall and bottom of the tube.
4. RNA elution
Remove the upper suspension. Wash the RNA pellet once with 75% ethanol. Add at least 1 ml of 75% ethanol for every 1 ml of TRIZOL. Mix the samples by vortexing and centrifuge at a high speed of no more than 7,500×g at 2-8°C for 5 minutes.
5. RNA re-dissolution
At the end of the operation, simply dry the RNA pellet. It is especially important that the RNA pellet cannot be completely dried as it will greatly reduce its solubility. The ratio of A260/280 for partially dissolved RNA samples is lt; 1.6. Use a pipette tip to pipette RNase-free water or 0.5% SDS solution several times to dissolve the RNA, and incubate at 55-60°C for 10 minutes.
三、Matters needing attention
1. The amount of sample and the amount of Trizol added must be in the above ratio. Do not increase the amount of sample or reduce the amount of Trizol arbitrarily. Otherwise, the endogenous RNase will be incompletely inhibited, leading to RNA degradation.
2. Change gloves in time during the whole process, wear a double-layer mask, and turn on the alcohol lamp in the operating area.
3. RNA must be stored in a refrigerator at -80°C, and the storage time at -20°C is very short.
4. The prepared solution should be sterilized DEPC water.
5. Glassware can be baked in an oven at 150°C for 4 hours. Plastic utensils can be soaked in 0.5 M NaOH for 10 minutes, rinsed thoroughly with water, and autoclaved for later use.