TRIzol RNA Extraction Protocol

For samples designated for RNA microarrays and RNA sequencing, relying solely on TRIzol for total RNA isolation is not recommended. TRIzol use often introduces protein and organic contaminants, potentially impeding the library production process. This caution is particularly relevant for small RNA sequencing (such as microRNA sequencing) and whole transcriptome sequencing, where the choice of TRIzol as the extraction reagent can lead to significant microRNA loss and the introduction of unwanted protein and organic impurities, negatively impacting subsequent library preparation.

Our recommended approach involves initiating isolation with TRIzol for optimal yields. Subsequently, achieving enhanced RNA purity is facilitated through additional purification steps, such as column purification or magnetic bead purification. This purified RNA is then deemed suitable for microarrays and RNA sequencing.

CD Genomics is dedicated to delivering personalized RNA sequencing solutions, from library construction to sequencing and bioinformatics analysis. If you have any queries regarding samples or RNA extraction, feel free to reach out to our technical team. Alternatively, explore our Sample Submission Guidelines for further guidance.

Recommended Reading:

A Guide to RNA Extraction, RNA Purification and Isolation

Troubleshooting Guide RNA Extraction for Sequencing

Principle of TRIzol RNA Extraction

The TRIzol reagent facilitates the direct extraction of total RNA from tissues or cells, comprising guanidine isothiocyanate, phenol, 8-hydroxyquinoline, and β-mercaptoethanol. During sample lysis or homogenization, TRIzol efficiently disintegrates cells, inhibits cellular nucleases, and preserves RNA integrity. Following centrifugation with chloroform, the sample segregates into an aqueous phase containing RNA, and an organic phase. Precipitation of DNA and proteins from the aqueous phase is achieved, followed by isopropanol-induced protein precipitation to diminish RNA contamination. Subsequently, ethanol is employed to precipitate intermediate DNA, ensuring further purification. The RNA precipitate is then reconstituted for subsequent applications, ensuring a reliable backup for downstream analyses.

What Is TRIzol Reagent?

(1) Guanidine isothiocyanate (Component I of TRIzol reagent): This potent uncoupling agent serves as a robust protein denaturant. It dissolves proteins, eliminates secondary protein structures, degrades cell structures, and facilitates the depolymerization of nucleic acid materials in cells, releasing RNA into the solution.

(2) Phenol and 8-hydroxyquinoline (Component II of TRIzol reagent): These components induce depolymerization of nuclear proteins and nucleic acids (chloroform can also achieve this). Phenol effectively denatures proteins, and the addition of 0.1% 8-hydroxyquinoline enhances inhibition of endogenous and exogenous RNases when used in conjunction with chloroform. Phenol not only disrupts cells to release RNA but also denatures and inactivates RNAase, thereby safeguarding RNA integrity.

(3) β-mercaptoethanol (Component III of TRIzol reagent): Primarily responsible for breaking disulfide bonds in RNase proteins.

(4) Chloroform: While contributing to protein denaturation, its primary function lies in phase separation. It expedites the stratification of the organic and aqueous phases, with RNA molecules remaining in the aqueous phase (pH 5.1), and DNA molecules precipitating at the phenol-solution interface. Additionally, chloroform can extract trace amounts of phenol from the nucleic acid solution.

(5) Isopropanol: Known for its compact volume and rapid action, it precipitates DNA and large RNA molecules, such as rRNA and mRNA. It selectively avoids precipitation of 5sRNA, tRNA, and polysaccharides. If the electrophoresis of RNA reveals unclear bands in the 5sRNA region, it may not necessarily indicate RNA degradation.

(6) Ethanol: Primarily employed for washing isopropanol, it also dissolves some proteins. The small amount used ensures easy evaporation.

Steps of RNA Extraction Using TRIzol

(1) Cell Culture

- Select cells in the logarithmic growth phase and inoculate them into a 6-well plate.

- Once the cell density exceeds 80%, add 500 µl TRIzol reagent (can be added after retrieval from a 4°C refrigerator).

- Mix thoroughly and perform repeated aspiration and agitation with a 1 ml tip.

- Allow it to stand at room temperature for 5 minutes to ensure complete separation of nucleic acid and protein complexes.

- For delayed RNA extraction, store the lysed sample at -80°C for long-term preservation.

(2) Tissue Homogenization

- Place an appropriate amount of tissue block into a 5 ml centrifuge tube, cut it into pieces, and add 1 ml TRIzol reagent per 50-100 mg of tissue.

- Ensure the tissue volume does not exceed 10% of the TRIzol volume.

- Homogenize the tissue thoroughly using an electric homogenizer to obtain the tissue homogenate.

(3) Chloroform Addition

- Add 200 µl of chloroform for every 1 ml of TRIzol reagent.

- Shake and mix for 15 seconds, then let it stand at room temperature for 2-3 minutes.

- Avoid using a vortexer to prevent genomic DNA breakage.

(4) Centrifugation

- Centrifuge the sample at 4°C, 12,000g/min for 15 minutes.

- The sample will separate into three layers: the bottom yellow organic phase, the upper colorless aqueous phase, and an intermediate layer. RNA predominantly exists in the upper aqueous phase.

- Collect the upper aqueous phase into a new RNase-free 1.5 ml centrifuge tube. Do not aspirate the intermediate layer.

- Store the lower organic phase at 4°C if both DNA and protein extraction are intended.

(5) Isopropanol Precipitation

- Add an equal volume of isopropanol to the centrifuge tube, mix gently by inverting, and let it stand at room temperature for 5-10 minutes.

(6) Centrifugation

- Centrifuge at 4°C, 12,000g/min, for 10 minutes to precipitate RNA at the bottom or wall of the tube.

(7) Wash with Ethanol

- Discard the supernatant, add 500 µl of pre-cooled 75% ethanol, mix well by gentle pipetting, and wash the precipitate.

(8) Centrifugation

- Centrifuge at 4°C, 12,000g/min for 5 minutes.

(9) Air Drying

- Discard the supernatant and allow the RNA pellet to air dry for 3-5 minutes on an ultra-clean surface, observing the degree of alcohol volatilization.

- Ensure the RNA samples are not excessively dry (control time < 5 minutes) to facilitate dissolution.

(10) Dissolution

- Add an appropriate amount of DEPC water (RNase Free) to dissolve RNA based on the extracted RNA concentration (typically add 20 µl DEPC water).

Schematic showing the standard TRIzol RNA extraction procedures shown on the left with modifications required. (Lee et al., 2011)

RNA Concentration and Purity Measurement

Principle

• The benzene ring structure of RNA bases exhibits strong ultraviolet absorption, with the highest peak at 260 nm.
• Proteins display multiple absorption peaks, reaching their maximum at 280nm, while the absorbance at 230 nm reflects the presence of ethanol, GTC, GUHCL, EDTA, etc.
• An OD260 value of 1 corresponds to 40 µg/ml single-stranded RNA, enabling the calculation of RNA sample concentration.
• The OD260/OD280 ratio provides an estimate of RNA purity.

Measurement Method

• For OD260 values between 0.1 and 1.0: RNA concentration (µg/ml) = OD260 × dilution × 40 µg/ml.
• An OD260/OD280 ratio between 1.9 and 2.1 indicates satisfactory purity.
• OD260/OD280 < 1.9 suggests protein or genome residue.
• OD260/OD280 > 2.1 hints at partial RNA degradation.
• OD260/OD230 < 2.0 implies contamination by salt ions, organic solvents, etc.

Evaluation of RNA Integrity

A pristine RNA sample should manifest three distinct bands: 28sRNA, 18sRNA, and 5sRNA. Both the 18sRNA and 28sRNA bands should appear well-defined without any noticeable trailing, with the brightness of the 28sRNA band being twice as intense as that of the 18sRNA band. The absence of a clear band indicates severe RNA degradation, while the presence of a fluorescent zone in or near the spiked wells suggests DNA contamination. This method serves as a robust means to gauge the structural integrity of RNA samples.

Troubleshooting Guide: RNA Extraction for Sequencing may be a helpful article.

Embarking on the right path is pivotal for success, and in the realm of transcriptome sequencing, the quality of RNA significantly influences the outcome. Explore our Sample Submission Guidelines for comprehensive insights. Should you harbor an interest in our cutting-edge transcriptome products, do not hesitate to reach out to our proficient technical team. We are here to propel your research endeavors forward.

Reference:

1. Lee, Juliana Tsz Yan, Wai Hung Tsang, and King Lau Chow. "Simple modifications to standard TRIzol® protocol allow high-yield RNA extraction from cells on resorbable materials." Journal of Biomaterials and Nanobiotechnology 2.01 (2011): 41.