The cellular RNAase should be inactivated as soon as possible during the first stage of the extraction process. Once the endogenous RNAase is destroyed, the possibility of RNA damage is greatly reduced and the purification process can proceed at a suitable pace. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Purification of RNA from tissues and cells by acid phenol-guanidine thiocyanate-chloroform extraction
Principle
The cellular RNAase should be inactivated as soon as possible during the first stage of the extraction process. Once the endogenous RNAase is destroyed, the potential for RNA damage is greatly reduced and the purification process can proceed at a suitable pace.
Materials and Instruments
Cells and tissues Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Ethanol Isopropanol Liquid nitrogen Phenol Phosphate buffer salt solution PBS Sodium acetate solution D (denaturing solution)
Colorimetric cups Tissue homogenizers Bars and mortars Polypropylene tubes Water baths
1. Solutions and buffers
Chloroform: isoamyl alcohol (49:1, V/V)
Ethanol
Isopropyl alcohol
liquid nitrogen
phenol
Phosphate buffered salt solution PBS (for suspension and monolayer cells only)
Sodium acetate (2 mol/L, pH 4.0)
Solution D (denaturing solution) 
Deionized formamide (optional)
Cells and tissues
2. Centrifugation and rotors
Sorvall SS-34 (or equivalent rotor); Sorvall H1000 (or equivalent rotor)
3. Specialized equipment
Colorimetric cup for measuring absorbance at 260 nm.
Tissue homogenizer
DEPC water-treated, pre-cooled rods and mortar and pestle
Polypropylene tubes
65°C water bath
Methods
1. Selection of suitable methods for extracting RNA from tissues or cells
(1) Tissue
(1) Tissue ① Isolate the tissue and place it immediately in liquid nitrogen.
② Place about 100 mg of frozen tissue in a mortar with liquid nitrogen and grind with a rod. Liquid nitrogen can be added during the grinding process to keep the tissue frozen.
(iii) Transfer the minced tissue into a tube containing 3 ml of Solution D.
④ Stir the tissue with a stirrer for 15-30 s at room temperature.
(2) Mammalian suspension cells
① Harvest cells by centrifugation at 200~1900 g for 5~10 min at room temperature (1000~3000 r/min with Sorvall RT600, H1000 rotor).
② Aspirate the culture medium and resuspend the cells in 1~2 ml of ice pre-cooled PBS.
③ Harvest the cells by centrifugation, thoroughly aspirate the PBS, and add 2 ml of solution D per 106 cells.
④ Stir the cells with a stirrer for 15-30 s at room temperature.
(3) Mammalian monolayer culture cells
① Aspirate the culture medium and wash the cells once with 5~10 ml of ice-cold PBS.
② Aspirate PBS and add Solution D to cover the cells, add 2 ml to 90 mm dish (1 ml to 60 mm dish).
③ Transfer the cell lysate to a tube.
③ Transfer the cell lysate to the tube. ④ Stir the lysed cells with a stirrer for 15~30 s at room temperature.
2. Transfer the mixture to a tube and immediately add 0.1 ml of 2 mol/L sodium acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol to each ml of Solution D. After each component is added, cap the tube and invert to mix. After adding each component, cap the tube and invert to mix.
3. The homogenate was shaken vigorously for 10 s. An ice bath was used for 15 min to allow complete cleavage of the nuclear protein complex.
4. 10,000 g (9000 r/min with Sorvall SS-34 rotor) centrifuge at 4°C for 20 min, and transfer the upper aqueous RNA-containing phase into a new tube.
5. Add an amount of isopropanol equal to the extracted RNA. Mix the liquid thoroughly and precipitate the RNA at -20°C for 1 h or longer.
6. Centrifuge at 10,000 g (9000 r/min with Sorvall SS-34 rotor) for 30 min at 4°C and collect the precipitated RNA.
7. Gently pour isopropanol into Solution D to dissolve the RNA particles, adding 0.3 ml of Solution D for every 1 ml of solution used in the first step.
8. Transfer the solution to a microcentrifuge tube, vortex to mix, and precipitate the RNA with an equal amount of isopropanol at -20°C for 1 h or longer.
9. Collect the RNA precipitate by centrifugation in a microcentrifuge at 4°C for 10 min at maximum speed. Wash the precipitate twice with 75% ethanol and repeat the centrifugation. Remaining ethanol is aspirated with a single-use pipette tip. Leave the uncapped tube on the bench for a few minutes to allow the ethanol to evaporate. the RNA should not be completely dry.
10. Add 50-100 μl of DEPC-treated water. Store the RNA solution at -70 °C.
11. Estimate the concentration of RNA. 
