Introduction to RNA Isolation Using the TRIzol Method
The TRIzol method is one of the most widely used techniques for isolating high-quality total RNA from biological samples. It is based on the acid guanidinium thiocyanate-phenol-chloroform extraction method developed by Chomczynski and Sacchi in 1987, which remains a gold-standard approach in molecular biology.
This method enables the rapid, efficient, and reliable extraction of total RNA from a wide range of biological materials, including cultured cells, animal tissues, plant tissues, and microorganisms.
During RNA isolation, samples are lysed in TRIzol reagent, a monophasic solution containing guanidinium thiocyanate and phenol. These chemicals disrupt cell and nuclear membranes, denature proteins, irreversibly inactivate RNases, and protect RNA from degradation.
After cell lysis, chloroform is added, and the mixture is centrifuged to separate it into three phases: an upper aqueous phase containing RNA, an interphase containing DNA, and a lower organic phase containing proteins, lipids, and other cellular components.
RNA is selectively recovered from the aqueous phase, allowing it to be effectively separated from DNA and proteins before further purification.
Commercial reagents such as TRIzol™ Reagent (Thermo Fisher Scientific) and TRI Reagent® (Sigma-Aldrich) have standardized this chemistry, providing improved reproducibility, consistent RNA yields, and greater accessibility across research laboratories.
TRIzol-based RNA isolation is widely used in molecular biology, genetics, biotechnology, and biomedical research because it produces high-quality, intact RNA suitable for downstream applications such as RT-PCR, RT-qPCR, RNA sequencing (RNA-Seq), gene expression analysis, transcriptomics, and Northern blotting.
Due to its high RNA yield, excellent RNA integrity, broad sample compatibility, and reliable performance, the TRIzol method continues to be one of the most trusted RNA extraction techniques used in research laboratories worldwide.
Key Reagents of the RNA isolation using the TRIzol method
TRIzol™ Reagent / TRI Reagent®: Approximately 1 mL per 50–100 mg of tissue or 1 × 10⁶ cultured cells. It lyses cells, denatures proteins, irreversibly inactivates RNases, and preserves RNA integrity during extraction.
Chloroform: 0.2 mL per 1 mL of TRIzol reagent. It induces phase separation during centrifugation, allowing RNA to partition into the upper aqueous phase while DNA and proteins remain in the interphase and organic phase.
Isopropanol: 0.5–1 mL. It precipitates RNA from the aqueous phase by reducing its solubility, enabling RNA pellet formation after centrifugation.
75% Ethanol: Prepared using RNase-free (DEPC-treated) water. It washes the RNA pellet to remove residual salts, phenol, and other contaminants while maintaining RNA integrity.
DEPC-treated Water / RNase-free Water: Used as required to dissolve and resuspend the purified RNA, making it suitable for storage and downstream applications such as RT-PCR, RT-qPCR, and RNA sequencing.
Principle of the RNA Isolation Using the TRIzol Method
The TRIzol method is based on the selective solubility and phase partitioning of biomolecules under acidic conditions, enabling the efficient separation of RNA from DNA, proteins, and other cellular components.
Guanidinium thiocyanate, a strong chaotropic agent present in TRIzol reagent, disrupts hydrogen bonding, denatures proteins, and irreversibly inactivates RNases and DNases, thereby protecting RNA from enzymatic degradation (Chomczynski & Sacchi, 1987).
Phenol assists in protein denaturation, dissolves lipids, and facilitates the breakdown of cellular and nuclear membranes, ensuring complete cell lysis and release of nucleic acids.
After cell lysis, chloroform is added, and the sample is centrifuged, causing the mixture to separate into three distinct phases based on differences in solubility and density.
The upper aqueous phase contains RNA because RNA remains soluble under the acidic conditions created by the extraction reagent.
The interphase contains most of the DNA, while the lower organic phase contains proteins, lipids, and other hydrophobic cellular components.
The RNA-containing aqueous phase is carefully transferred to a new tube, where isopropanol is added to reduce RNA solubility, causing it to precipitate and form a visible RNA pellet after centrifugation.
The RNA pellet is subsequently washed with 75% ethanol to remove residual salts, phenol, and other contaminants, improving RNA purity.
Finally, the purified RNA pellet is air-dried briefly and dissolved in RNase-free (DEPC-treated) water, producing high-quality RNA suitable for downstream applications such as RT-PCR, RT-qPCR, RNA sequencing (RNA-Seq), Northern blotting, and gene expression analysis.
Steps / Protocol of the RNA isolation using the TRIzol method
1. Cell Culture
Select healthy cells in the logarithmic (log) growth phase and seed them into a 6-well culture plate.
When the cell confluency reaches approximately 80%, add 500 µL of TRIzol reagent directly to each well.
Pipette the lysate up and down several times using a 1 mL pipette tip to ensure complete cell lysis and thorough mixing.
Incubate the lysate at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes.
If RNA extraction cannot be performed immediately, store the lysed samples at −80°C for long-term preservation.
2. Tissue Homogenization
Place 50–100 mg of fresh or frozen tissue into a 5 mL RNase-free centrifuge tube and cut it into small pieces.
Add 1 mL of TRIzol reagent for every 50–100 mg of tissue.
Ensure that the tissue volume does not exceed 10% of the TRIzol reagent volume for efficient lysis.
Homogenize the tissue thoroughly using an electric or mechanical homogenizer until a uniform homogenate is obtained.
3. Chloroform Addition
Add 200 µL of chloroform for every 1 mL of TRIzol reagent used.
Securely cap the tube and shake vigorously by hand for 15 seconds to mix thoroughly.
Allow the mixture to stand at room temperature for 2–3 minutes.
Avoid vortexing the sample, as excessive mechanical force may damage nucleic acids.
4. Phase Separation by Centrifugation
Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.
After centrifugation, three distinct layers will form: the upper colorless aqueous phase (RNA), the interphase (DNA), and the lower organic phase (proteins and lipids).
Carefully transfer only the upper aqueous phase into a new RNase-free 1.5 mL microcentrifuge tube, avoiding disturbance of the interphase.
If DNA or protein isolation is also required, retain the lower phases for subsequent extraction.
5. RNA Precipitation with Isopropanol
Add an equal volume of isopropanol to the collected aqueous phase.
Mix gently by inverting the tube several times until the solution is homogeneous.
Incubate the mixture at room temperature for 5–10 minutes to allow RNA precipitation.
6. RNA Pellet Formation
Centrifuge the sample at 12,000 × g for 10 minutes at 4°C.
A white or translucent RNA pellet will form at the bottom or along the side of the tube.
7. Washing the RNA Pellet
Carefully discard the supernatant without disturbing the RNA pellet.
Add 500 µL of pre-chilled 75% ethanol to wash the pellet.
Gently mix by slowly pipetting up and down or by inverting the tube.
8. Ethanol Wash Centrifugation
Centrifuge the sample at 12,000 × g for 5 minutes at 4°C.
Carefully remove and discard the ethanol without disturbing the RNA pellet.
9. Air Drying
Air-dry the RNA pellet for 3–5 minutes on a clean RNase-free surface.
Avoid over-drying the pellet (generally less than 5 minutes), as excessively dry RNA becomes difficult to dissolve.
10. RNA Dissolution
Dissolve the RNA pellet in an appropriate volume of RNase-free (DEPC-treated) water, typically 20 µL, depending on the expected RNA yield.
Gently pipette up and down or incubate for a few minutes to ensure complete dissolution before quantification or downstream molecular applications.
Observations and Results
Successful RNA isolation using the TRIzol method is indicated by the formation of a small, white or translucent RNA pellet after isopropanol precipitation and centrifugation.
The RNA pellet may appear gel-like or loosely attached to the bottom or side of the centrifuge tube, particularly when extracting high-molecular-weight RNA, and should be handled carefully to avoid sample loss (Sigma-Aldrich, n.d.).
The purified RNA should dissolve readily in RNase-free (DEPC-treated) water, producing a clear solution without visible contaminants.
When analyzed by agarose gel electrophoresis, high-quality eukaryotic RNA typically shows two sharp and distinct ribosomal RNA bands (28S and 18S rRNA) with little or no smearing, indicating that the RNA is intact.
A 28S:18S rRNA band intensity ratio of approximately 2:1 is generally considered an indicator of good RNA integrity in eukaryotic samples.
Excessive smearing, faint ribosomal RNA bands, or the absence of distinct bands usually indicates RNA degradation, which may result from RNase contamination, harsh mechanical mixing, prolonged sample handling, or improper storage.
Poor phase separation or accidental transfer of the interphase during extraction may result in DNA or protein contamination, reducing RNA purity and affecting downstream applications.
High-quality RNA obtained using the TRIzol method is suitable for downstream molecular techniques such as RT-PCR, RT-qPCR, RNA sequencing (RNA-Seq), Northern blotting, cDNA synthesis, and gene expression analysis.
Modifications of the RNA Isolation Using the TRIzol Method
Adjusting TRIzol volume according to sample type: For cell culture samples with low biomass, the volume of TRIzol reagent can be reduced while maintaining the recommended TRIzol-to-cell ratio. This minimizes reagent dilution and organic solvent carryover, resulting in a higher RNA concentration without affecting cell lysis or RNase inactivation (CD Genomics, 2023; Thermo Fisher Scientific, 2024).
Additional chloroform extraction for lipid-rich tissues: Samples with high lipid or membrane content, such as adipose tissue or brain tissue, may benefit from an additional chloroform extraction step. This improves phase separation, reduces phenol and lipid contamination, and increases the purity of the isolated RNA (Sigma-Aldrich, n.d.; Bitesize Bio, 2024).
Gentle mixing instead of vortexing: During chloroform addition, gently inverting the tube instead of vortexing minimizes mechanical stress and helps preserve RNA integrity. This modification is particularly important for applications requiring high-quality RNA, such as gene expression analysis and RNA sequencing (Chomczynski & Sacchi, 1987; Thermo Fisher Scientific, 2024).
Performing centrifugation at 4°C: Carrying out all centrifugation steps at 4°C helps suppress residual RNase activity, improves RNA stability, and enhances the efficiency of phase separation and RNA precipitation. This is especially beneficial when processing RNase-rich or temperature-sensitive samples (Thermo Fisher Scientific, 2024; Bitesize Bio, 2024).
Using glycogen or linear acrylamide as a carrier (optional): When extracting RNA from samples with very low RNA content, adding a carrier such as glycogen or linear acrylamide during isopropanol precipitation can improve RNA pellet visibility and increase RNA recovery without interfering with most downstream applications (Thermo Fisher Scientific, 2024).
Incorporating an on-column or enzymatic DNase treatment: For applications requiring highly pure RNA, such as RT-qPCR or RNA sequencing, a DNase treatment can be performed after RNA isolation to remove residual genomic DNA and improve the accuracy of downstream analyses (Thermo Fisher Scientific, 2024).
Troubleshooting of the RNA Isolation Using the TRIzol Method
Problem: Low RNA yield
Likely Cause: Incomplete cell or tissue lysis, insufficient homogenization, or inadequate sample disruption.
Solution: Ensure complete homogenization of the sample, use the recommended volume of TRIzol reagent, and allow the lysate to incubate for 5 minutes at room temperature before phase separation.
Problem: RNA degradation
Likely Cause: RNase contamination or improper sample handling.
Solution: Use RNase-free tubes, pipette tips, and reagents, wear gloves, clean work surfaces with RNase decontamination solution, and keep samples cold whenever possible.
Problem: Phenol contamination
Likely Cause: Incomplete phase separation or accidental transfer of the interphase or organic phase during RNA collection.
Solution: Carefully transfer only the upper aqueous phase without disturbing the interphase. If necessary, perform an additional chloroform extraction before RNA precipitation.
Problem: Low A260/A280 ratio
Likely Cause: Protein or phenol contamination.
Solution: Wash the RNA pellet thoroughly with 75% ethanol, repeat the ethanol wash if necessary, and avoid transferring contaminants during phase separation.
Problem: No visible RNA pellet
Likely Cause: Low cell or tissue input, low RNA concentration, or loss of the pellet during washing.
Solution: Increase the starting sample amount when possible, add a carrier such as glycogen or linear acrylamide during isopropanol precipitation, and carefully handle the pellet during washing and drying.
Quality Assessment of the Isolated RNA
A260/A280 Ratio: A spectrophotometric absorbance ratio of approximately 1.9–2.1 indicates high RNA purity with minimal protein contamination and effective removal of phenol residues. RNA within this range is generally considered suitable for sensitive downstream applications, including enzymatic reactions and transcriptomic analyses.
A260/A230 Ratio: An absorbance ratio greater than 2.0 suggests efficient removal of organic contaminants, salts, and chaotropic agents such as guanidinium thiocyanate. A high A260/A230 value indicates minimal carryover of extraction reagents that could interfere with downstream molecular techniques.
Gel Electrophoresis Analysis: Intact RNA typically produces sharp and well-defined ribosomal RNA (rRNA) bands with minimal smearing on agarose gels. The presence of clear 28S and 18S rRNA bands indicates successful RNase inactivation, proper sample handling, and low RNA degradation.
Absence of Phenol Odor: A properly purified RNA sample should have little to no detectable phenol smell. The absence of a strong phenol odor indicates effective removal of organic solvents during washing steps, reducing the possibility of enzymatic inhibition and improving the accuracy of RNA quantification and amplification-based assays.
Overall RNA Quality: High-quality RNA should demonstrate appropriate purity ratios, intact ribosomal RNA patterns, and minimal contamination, ensuring reliable performance in downstream applications such as RT-PCR, RT-qPCR, RNA sequencing (RNA-Seq), cDNA synthesis, and gene expression studies.
Safety Tips and Precautions of RNA Isolation Using The TRIzol Method
Handle phenol-containing reagents in a chemical fume hood: TRIzol and other phenol-based reagents contain hazardous chemicals such as phenol and chloroform, which are toxic, corrosive, and volatile. All procedures involving these reagents should be performed inside a certified chemical fume hood to minimize inhalation risks and prevent accidental exposure (Thermo Fisher Scientific, 2024).
Wear appropriate personal protective equipment (PPE): Always use suitable laboratory protection, including nitrile gloves, a fully closed lab coat, and safety goggles, to protect against chemical exposure, eye injury, and contamination while handling hazardous reagents and biological samples.
Prevent RNase contamination: Use RNase-free consumables, sterile pipette tips, and clean working surfaces throughout the procedure. Wear gloves and change them frequently to prevent RNase transfer from hands, which can degrade RNA samples.
Handle biological samples carefully: Treat all biological materials as potentially hazardous and follow appropriate biosafety practices during sample collection, processing, and disposal.
Dispose of chemical waste properly: Waste containing phenol, chloroform, and other organic solvents must be collected separately in designated hazardous waste containers and disposed of according to institutional guidelines and environmental safety regulations.
Avoid direct contact with TRIzol reagents: In case of accidental exposure, immediately follow laboratory safety procedures, including washing affected areas with appropriate amounts of water and reporting the incident according to institutional protocols.
Maintain a clean and organized workspace: Perform RNA isolation in an RNA-safe environment, minimize unnecessary handling, and keep reagents properly stored to maintain RNA quality and ensure reproducible results.
Storage and Long‑Term Stability of the Isolated RNA
Short-term storage at 4°C: Purified RNA can be stored at 4°C for up to 24 hours when immediate downstream applications, such as cDNA synthesis or RT-PCR, are planned. Samples should be protected from RNase contamination during handling and storage.
Long-term storage at −80°C: For extended storage, RNA samples should be maintained at −80°C to preserve RNA integrity by reducing hydrolytic degradation and minimizing residual RNase activity. This storage condition is recommended for archived samples and delayed applications, including transcriptomic analysis and RNA sequencing.
Avoid repeated freeze–thaw cycles: Multiple freeze–thaw cycles can cause RNA fragmentation and reduce sample quality. To maintain RNA stability and ensure reproducible experimental results, RNA samples should be divided into smaller aliquots before long-term storage.
Use RNase-free storage conditions: RNA samples should be stored in RNase-free tubes and handled using RNase-free materials to prevent degradation during storage.
Applications of RNA Isolation Using the TRIzol Method
RT-PCR and qRT-PCR: High-quality RNA extracted using the TRIzol method is suitable for reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR), enabling accurate detection and measurement of specific gene expression levels.
RNA Sequencing (RNA-Seq) and Transcriptome Analysis: Intact RNA obtained through TRIzol extraction is widely used in next-generation sequencing workflows. It allows comprehensive analysis of transcript abundance, alternative splicing patterns, transcript variations, and differential gene expression profiles.
Gene Expression Profiling: TRIzol-isolated RNA provides reliable material for comparing gene expression patterns among different tissues, developmental stages, experimental conditions, or disease states, supporting both targeted and large-scale transcriptional studies.
Northern Blotting: RNA extracted using the TRIzol method maintains sufficient integrity and molecular size distribution for Northern blot analysis, allowing detection, characterization, and size estimation of specific RNA transcripts.
cDNA Synthesis: Purified RNA serves as an essential template for complementary DNA (cDNA) synthesis, which is required for downstream applications such as molecular cloning, PCR amplification, gene expression analysis, and recombinant DNA studies.
Molecular and Biomedical Research Applications: Due to its ability to produce high-quality total RNA, the TRIzol method is widely applied in genomics, molecular biology, biotechnology, disease research, and functional gene studies.
Advantages of RNA Isolation Using the TRIzol Method
High RNA yield and integrity: The TRIzol method efficiently extracts total RNA while maintaining transcript integrity through rapid RNase inactivation and effective disruption of cellular and nuclear structures.
Effective RNase inactivation: The combination of guanidinium thiocyanate and phenol rapidly denatures and inactivates RNases, protecting RNA from degradation during cell lysis and extraction, even when working with RNase-rich tissues.
Cost-effective and widely validated: TRIzol-based RNA isolation is an economical alternative to many column-based extraction methods and has been extensively validated across a wide range of biological samples and research applications.
Compatible with downstream molecular applications: Properly purified RNA obtained using the TRIzol method is suitable for various molecular techniques, including reverse transcription, PCR amplification, quantitative PCR, RNA sequencing, and other transcriptomic analyses, without significant interference from extraction reagents.
Broad sample compatibility: The method can be applied to diverse biological materials, including cultured cells, animal tissues, plant samples, and microorganisms, making it a versatile approach for RNA extraction.
Limitations of the RNA isolation using the TRIzol method
Use of hazardous chemicals: The TRIzol method requires the use of phenol and chloroform, which are toxic, corrosive, and volatile chemicals. Therefore, strict safety measures, proper handling procedures, appropriate chemical waste disposal, and trained laboratory personnel are required.
Labor-intensive compared to spin-column methods: Unlike commercial spin-column RNA extraction kits, the TRIzol method involves multiple manual steps, including cell/tissue lysis, phase separation, RNA precipitation, washing, and resuspension. These additional steps increase processing time and may introduce variation between samples.
Risk of phenol and organic solvent contamination: Incomplete removal of phenol, chloroform, or other organic contaminants during extraction can negatively affect RNA purity and interfere with downstream enzymatic applications such as reverse transcription, PCR, and RNA sequencing. Careful phase separation and thorough ethanol washing are essential to minimize contamination.
Possibility of genomic DNA contamination: Improper separation of the interphase during extraction may result in carryover of genomic DNA, which can interfere with gene expression studies and other RNA-based analyses.
Lower suitability for high-throughput applications: Due to its manual nature and requirement for multiple handling steps, TRIzol extraction is less suitable for large-scale sample processing compared with automated or column-based RNA isolation platforms.
Conclusion
RNA isolation using the TRIzol method remains a robust, versatile, and widely validated technique for extracting high-quality RNA from diverse biological samples.
Based on the foundational acid guanidinium thiocyanate-phenol-chloroform extraction method developed by Chomczynski and Sacchi (1987), TRIzol extraction continues to be extensively used in molecular biology due to its ability to simultaneously recover RNA, DNA, and proteins from a single biological sample.
Despite limitations associated with the use of hazardous reagents, manual processing steps, and the requirement for careful handling, optimized protocols and proper laboratory safety practices ensure consistent RNA yield, purity, and integrity.
The high-quality RNA obtained through the TRIzol method supports a wide range of downstream applications, including gene expression analysis, RT-PCR, qRT-PCR, RNA sequencing, transcriptomics, and molecular diagnostics.
Due to its reliability, cost-effectiveness, and broad applicability, TRIzol-based RNA extraction remains an essential and widely adopted technique in molecular biology, biotechnology, and biomedical research.
References
Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry, 162(1), 156–159. https://doi.org/10.1016/0003-2697(87)90021-2
Thermo Fisher Scientific. (2024). TRIzol™ Reagent RNA Isolation Protocol and Troubleshooting Guide. Thermo Fisher Scientific. Available at: https://www.thermofisher.com/np/en/home/references/protocols/nucleic-acid-purification-and-analysis/mrna-protocols/trizol-plus-rna-purification-kit.html
CD Genomics. (2023). TRIzol RNA Extraction Protocol. CD Genomics. Available at: https://rna.cd-genomics.com/resource/trizol-protocol.html
Sigma-Aldrich. (n.d.). TRI Reagent® Protocol: RNA Isolation Using Acid Guanidinium Thiocyanate–Phenol–Chloroform Extraction. Available at: https://www.sigmaaldrich.com/NP/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/tri-reagent
Yeasen Biotechnology. (2025). Common Issues and Troubleshooting in RNA Extraction Using TRIzol. Yeasen Biotech. Available at: https://www.yeasenbio.com/blogs/molecular-biology/common-issues-and-troubleshooting-in-rna-extraction-using-trizol
Bitesize Bio. (2024). Troubleshooting RNA Isolation: Common Problems and Solutions. Bitesize Bio. Available at: https://bitesizebio.com/2345/troubleshooting-rna-isolation/
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