Molecular Biology-RNA Extraction-Trizol Method-Guanidine thiocyanate

Experimental principle

The main substance of Trizol is guanidine isothiocyanate, which can destroy the cell and release the RNA while protecting the integrity of the RNA. After adding chloroform and centrifuging, the sample is separated into a water layer and an organic layer. RNA is present in the water layer. After collecting the upper water layer, RNA can be reduced by isopropanol precipitation. Whether it is human, animal, plant or bacterial tissue, the Trizol method is good for a small amount of tissue (50-100 mg) and cells (5×106) and a large number of tissues (≧1 g) and cells (>107) seperate effect. The simplicity of Trizol reagent operation allows multiple samples to be processed simultaneously. All operations can be completed within an hour. The total RNA extracted by Trizol can avoid DNA and protein contamination. Therefore, it can be used for Northern blot analysis, dot blotting, poly(A) selection, in vitro translation, RNase protection analysis and molecular cloning.

Main reagent

Trizol solution, chloroform, isopropanol, 75% ethanol, DEPC

Major equipment

Ultra-clean workbench, 1.5 mL centrifuge tube, pipette, UV spectrophotometer, quartz cuvette, swimming tank and mold, electrophoresis instrument, gel imaging system

Experimental Materials

Escherichia coli

Experimental steps

  1. Use Trizol solution to extract bacterial total RNA

(1) Pick a single colony of engineering bacteria, culture it to the stable stage, take 3 mL of the bacterial solution and centrifuge to obtain the bacteria in a 1.5 mL centrifuge tube;

(2) Add 1 mL of Trizol solution to each tube, cap the tube tightly, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes;

(3) Centrifuge at 12000 g at 4°C for 10 min;

(4) Take the supernatant and transfer (about 1 mL) to a new 1.5 mL centrifuge tube;

(5) Add 0.2 mL of chloroform (0.2 volume Trizol) to each tube, close the cap tightly, and shake vigorously for 15 s;

(6) Let stand at room temperature for 3 min;

(7) Centrifuge for 10 min at 4℃, 12000 g;

(8) Carefully aspirate the upper water phase, transfer it to another new 1.5 mL centrifuge tube, and measure its volume;

(9) Add 1 volume of chloroform, cover tightly, and shake vigorously for 15 s;

(10) Let stand at room temperature for 3 min;

(11) 4℃, 12000 g, centrifugation for 10 min;

(12) Carefully take the upper aqueous phase and transfer it to another 1.5 mL centrifuge tube that has been numbered;

(13) Add 0.5 mL of isopropanol (0.5 volume Trizol), gently invert and mix;

(14) At room temperature, let stand for 10 min;

(15) Centrifuge at 12000 g at 4°C for 10 min, and the RNA will sink to the bottom of the tube;

(16) Carefully aspirate the supernatant, add 1 mL of 75% ethanol (pre-cooled), and gently invert to wash the precipitate;

(17) Centrifuge at 7500 g at 4°C for 5 min;

(18) Carefully discard the supernatant, separate slightly, aspirate the remaining ethanol, and dry it at room temperature for 10 min;

(19) Dissolve each tube with 50 μL DEPC-treated double-distilled deionized water, incubate at 55-60°C for 5 minutes, and store at -80°C (storable for 5 weeks);

  1. Determination of RNA concentration

Dilute 10 µL of RNA sample with double-distilled water to 2 mL, transfer it to a quartz cuvette pre-soaked in absolute ethanol overnight and then dry it. Carefully remove air bubbles, use an equal volume of double-distilled water as a blank control. Measure the light absorption values of 260 nm, 280 nm, and 360 nm (reference wavelength) in the photometer, and calculate the RNA concentration according to the formula.

RNA concentration (µg/µL) = OD260 × dilution factor (200) × 40 (µg/mL)/1000

The OD260/OD280 ratio of the pure RNA sample is 1.8~2.0. If it is lower than this value, it indicates that there is protein contamination, and it can be extracted again with phenol/chloroform. When the value is 2.0, RNA purity is the highest.

  1. RNA quality testing

The RNA is detected by agarose gel electrophoresis, with the purpose of detecting the integrity of 28 S and 18 S bands and their ratio, or the integrity of mRNA smear. It is generally believed that if the 28 S and 18 S bands are bright and the edges are clear, and the brightness of 18 S is more than twice that of the 18 S band, the quality of RNA is better.

(1) Place the cleaned and dried electrophoresis tank and mold horizontally on the workbench;

(2) Accurately weigh 0.15 g of agarose and add it to 15 mL 1 × TAE. Shake well. Dissolve the agarose solution in the microwave oven in the shortest time (medium heat for 2 min). When it is cooled to 60℃, add GoldView (The final concentration is 5 µL/mL), mix well;

(3) Insert a suitable comb, pour the warm gel into the plastic mold, the thickness of the gel is 3~5 mm, and place it at room temperature to solidify (about 30 min);

(4) After the gel has solidified, carefully remove the comb, put the gel in the electrophoresis tank, and add electrophoresis buffer to make the liquid level 1~2 mm higher than the gel;

(5) Use a micropipette to mix the sample with the loading buffer and carefully add the mixture to the loading well, while adding a DNA molecular weight standard in the appropriate molecular weight range as a control;

(6) Cover the electrophoresis tank cover, adjust the voltage to 100 V, and electrophoresis for about 40 minutes;

(7) After the electrophoresis is completed, the gel is placed on the ultraviolet transmission detector to observe the results, and the gel imaging system is used to photograph and save.