TRIzol RNA Extraction Protocol

In the realm of molecular biology, the intricate dance between nucleic acids orchestrates the symphony of life. Among these, RNA emerges as a pivotal player, its multifaceted roles spanning from genetic regulation to cellular signaling. Understanding and extracting RNA with precision is paramount in unraveling the mysteries of biological processes. This comprehensive guide navigates through the nuances of TRIzol RNA extraction protocol, offering insights into its experimental principle, procedures, and precautions.

What is TRIzol?

TRIzol, also known as the isothiocyanate muscle-phenol-chloroform one-step method, represents a revolutionary approach to RNA extraction. This single-step method enables the direct isolation of total RNA from cells or tissues, preserving its integrity throughout the process. The amalgamation of isothiocyanate and phenol facilitates robust cell lysis while ensuring efficient separation of RNA from proteins and DNA.

Principles of RNA Extraction by Trizol

The TRIZOL reagent is a reagent for directly extracting total RNA from cells or tissues. It can maintain the integrity of RNA during cell lysis and dissolution. The main components of the reagent are thiocyanate and phenol. Thiocyanate can lyse cells, promote the dissociation of nucleoprotein complexes, separate RNA from proteins, and release RNA into the aqueous phase. After adding chloroform and centrifugation, the sample is divided into a colorless aqueous layer and a yellow organic layer. RNA is present in the aqueous layer. After collecting the aqueous layer, RNA can be recovered by precipitation with isopropanol. After removing the aqueous layer, DNA and proteins in the sample can also be sequentially recovered by precipitation. Ethanol precipitation can precipitate DNA from the intermediate layer, while adding isopropanol to the organic layer can precipitate proteins.

Steps for RNA Extraction by Trizol

Reagent Preparation

• Chloroform
• Isopropanol
• 75% Ethanol (in DEPC-treated water)
• RNase-free water or 0.5% SDS solution [Prepare RNase-free water by filling RNase-free glass bottles with water, adding diethylpyrocarbonate (DEPC) to a final concentration of 0.01% (v/v). Let it stand overnight, then autoclave. Prepare 0.5% SDS solution using DEPC-treated, autoclaved water].

Operational Processes

(1) Homogenize and lyse the samples

• For tissue samples: Add 1 ml of TRNsol per 10-100 mg of tissue. It is recommended to use a mechanical homogenizer for homogenization. Alternatively, tissues can be crushed using liquid nitrogen and transferred to a clean 1.5 ml microcentrifuge tube. Allow the powder to stand at 15-30°C for 5 minutes to thoroughly separate nucleoprotein complexes. The volume of the sample should not exceed 10% of the volume of TRNsol used, otherwise, DNA contamination may occur.
• For suspended cell cultures: Centrifuge and collect the cells. 1 ml of TRNsol reagent can be used to process (5~10) × 10^6 animal, plant, or yeast cells, or 1 × 10^8 bacterial cells. Avoid washing the cells before adding TRNsol, as this may increase the likelihood of mRNA degradation and RNA enzyme contamination. Mechanical homogenization or enzymatic digestion is typically required for plant, fungal, and yeast cells.
• For monolayer adherent cell cultures: Directly add 1 ml of TRNsol to a 3.5 cm diameter culture dish to lyse the cells. Use a pipette tip to pipette and break up the lysate several times. The amount of TRNsol used depends on the surface area of the culture dish (1 ml/10 ml). Insufficient TRNsol may result in DNA contamination in the separated RNA. If the lysate is viscous when aspirating with a pipette tip, additional TRNsol may be required.
• For blood samples: Add 250 μl of fresh whole blood to 750 μl of TRNsol, vigorously invert to mix. Note: In the second step, add 0.2 ml of chloroform.

(2) Phase separation

Add 0.2 ml of chloroform per 1 ml of TRNsol. Cover the centrifuge tube and vigorously shake for 15 seconds, then incubate on ice for 5 minutes. Centrifuge at 12,000 × g for 10 minutes at room temperature. At this point, the mixture separates into three layers: the bottom layer is the phenol-chloroform phase (red), the middle layer, and the upper aqueous phase (colorless). The aqueous phase accounts for 60% of the total TRIZOL volume, and RNA is completely present in the aqueous phase.

(3) RNA precipitation

Transfer no more than 80% of the upper aqueous phase to a new centrifuge tube, and add isopropanol to the aqueous phase. For every 1 ml of TRNsol used, add 500 μl of isopropanol (Note: For samples rich in polysaccharides or polyphenols, add 300 μl of isopropanol and 300 μl of 0.8 M sodium citrate/1.2 M NaCl mixture simultaneously). Vortex to mix thoroughly, then incubate at room temperature for 10 minutes. Centrifuge at 12,000 × g for 10 minutes at 4°C. Before centrifugation, flocculent gel-like precipitation can be observed on the side walls and bottom of the tube, which is RNA precipitation. For samples rich in sugars: such as polysaccharide-rich plant samples or samples containing glycoproteins and glycosaminoglycans, the following modified precipitation method is required to obtain pure RNA. Prepare Buffer A (1.2 M NaCl, 0.8 M sodium citrate). After completing the second step, immediately add isopropanol and Buffer A to the aqueous phase (add 0.3 ml of isopropanol and 0.3 ml of Buffer A for every 1 ml of TRNsol). Vortex to mix thoroughly, and centrifuge at room temperature, ≤12,000 × g for 10 minutes. This high-salt precipitation will reduce the co-purification of complex sugars.

(4) RNA washing

Discard the supernatant and wash the precipitate once with 75% ethanol. Vortex to mix the sample thoroughly, and centrifuge the sample at room temperature at a speed not exceeding 7,500 × g for 5 minutes. Note: 75% ethanol must be prepared with DEPC-treated sterile water, otherwise, residual RNaseA in the 75% ethanol will degrade RNA.

(5) RNA Dissolution

Carefully aspirate the ethanol, briefly centrifuge to pellet the droplets to the bottom of the tube, and carefully aspirate the remaining solution with a pipette. Air-dry the RNA at room temperature for 5-10 minutes. Do not use a centrifugal drying apparatus or vacuum drying apparatus, as excessive drying can make it difficult to redissolve RNA in water. Add appropriate DEPC-treated water to dissolve RNA.

(6) RNA Re-dissolution

Remove the supernatant and air-dry the RNA precipitate for 5-10 minutes in a vacuum or in air (do not centrifuge dry in a vacuum). Note: Do not completely dry the RNA precipitate, as this will greatly reduce its solubility. For partially dissolved RNA samples with an A260/280 ratio < 1.6.

Resuspend the RNA precipitate in RNase-free water or 0.5% SDS solution, pipette up and down several times, and let stand for 10 minutes at 55-60°C. After measuring the concentration, store at -20°C.

(7) Concentration Measurement

Take 2 μl of the storage solution and add it to another EP tube, then add 98 μl of RNase-free water, centrifuge to mix thoroughly, and use RNase-free water as a blank control to measure the OD260/280 value. 1 OD = 40 μg RNA. An OD260/280 value between 1.8-2.0 indicates high purity. Concentrations around 1000 ng/ml are more accurate. If the concentration is too high, dilute before re-measuring.

(8) RNA Identification

Perform formaldehyde denaturing agarose gel electrophoresis to determine the integrity of the extracted RNA and the presence of DNA contamination.

Notes for Trizol

• When using Trizol, it is essential to wear gloves and goggles to avoid skin and clothing contact and to use a fume hood to prevent inhalation of vapors.
• For extracting RNA from small samples (1-10 mg tissue or 10²-10⁴ cells), a small amount of glycogen can be added to promote RNA precipitation. For example, when homogenizing samples with 800 ml of TRIzol, adding 5-10 μg of RNase-free glycogen before RNA precipitation is recommended. Glycogen will precipitate along with RNA, and a glycogen concentration not exceeding 4 mg/ml does not affect the synthesis of the first strand or PCR reactions.
• Samples homogenized before adding chloroform can be stored at -60 to -70°C for at least one month. RNA precipitate can be stored in 75% alcohol at 2-8°C for more than a week or at -5 to -20°C for more than a year.
• During layering and RNA precipitation, a benchtop centrifuge can also be used, with centrifugation at 2600 × g for 30-60 minutes.
• Expected yields for RNA extraction from 1 mg of tissue or 1 × 10⁶ cells are as follows:

TRIzol RNA Extraction Protocol

- mg g kg ton

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