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Who this is for: researchers and lab staff who need to understand what TRIzol reagent does, why its phases separate the way they do, and which numbers in the manufacturer’s guide matter. General laboratory information only; follow your own SOP and the manufacturer’s current user guide at the bench.
Short answer
TRIzol RNA extraction is a single-step liquid-phase method. TRIzol Reagent, a monophasic solution of phenol and guanidine isothiocyanate, lyses the sample and inactivates RNases. Adding chloroform and centrifuging splits the homogenate into an upper aqueous phase that holds the RNA, an interphase, and a lower organic phase that holds DNA and protein. The RNA is then precipitated from the aqueous phase with isopropanol. The manufacturer states the reagent is for research use only, not for diagnostic procedures.
What TRIzol Reagent is
Invitrogen’s TRIzol Reagent User Guide (Pub. No. MAN0001271 Rev. D) describes the product as a “monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components”. It is designed to isolate total RNA, and also DNA and protein, from cell and tissue samples of human, animal, plant, yeast or bacterial origin, in about an hour. The guanidine isothiocyanate and phenol disrupt cells while strongly inhibiting RNase activity, which is what keeps the RNA intact during homogenisation.
The chemistry is a branded implementation of the acid guanidinium thiocyanate-phenol-chloroform method published by Chomczynski and Sacchi in Analytical Biochemistry in 1987. Their abstract describes a single extraction that gives “a pure preparation of undegraded RNA in high yield”, completed within 4 hours, and is “particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.” Other suppliers sell equivalent reagents under generic names such as TRI Reagent; the principle is the same, but parameters should always come from the guide that ships with the specific product you use.
What does TRIzol do in RNA extraction?
It does two jobs at once: it breaks open cells and it protects RNA from degradation. Because RNases are tough and ubiquitous, inactivating them in the very first step (before the sample is handled further) is what makes the method reliable. Chloroform is not the lysing agent; its role is to drive separation of the homogenate into distinct phases so that RNA, DNA and protein end up in different layers.
How the three fractions separate
After homogenisation, chloroform addition and centrifugation, the manufacturer’s guide describes three layers. The table below restates that layout and the precipitant the guide names for each fraction.
| Layer after centrifugation | Appearance (per guide) | Contains | Recovered by precipitating with |
|---|---|---|---|
| Upper aqueous phase | Clear | RNA | Isopropanol |
| Interphase | Between the layers | DNA (with the organic phase) | Ethanol |
| Lower organic phase | Red | DNA and proteins | Ethanol (DNA); isopropanol from the phenol-ethanol supernatant (protein) |
The same lysate can therefore yield RNA, DNA and protein from one sample, which is a practical reason labs choose the reagent over RNA-only column workflows. The interphase and organic layer also explain a common technical point: genomic DNA is physically present in the lower fractions, so careful removal of the aqueous phase (the guide specifies transferring it with the tube angled at 45 degrees, avoiding the interphase) is what limits DNA carry-over. The guide itself does not describe a DNase step; for DNase treatment of extracted RNA see RNA extraction protocol basics.
Parameters scaled per 1 mL of TRIzol
Everything in the guide scales from the volume of TRIzol used. This sheet collects the stated values so a scaled-up or scaled-down run can be checked at a glance. It is a reference for understanding the method, not a replacement for the current guide.
| Stage | Value per 1 mL TRIzol (Rev. D guide) |
|---|---|
| Sample input, tissue | 50-100 mg |
| Sample input, monolayer cells | 1 x 105 to 1 x 107 cells (3.5 cm dish) |
| Sample input, suspension cells | 5-10 x 106 animal, plant or yeast cells; 1 x 107 bacterial cells |
| Maximum sample volume | Not more than 25% of the TRIzol volume |
| Chloroform | 0.2 mL |
| Phase separation spin | 15 min, 12,000 x g, 4 °C |
| Isopropanol | 0.5 mL; 10 min at room temperature |
| Precipitation spin | 10 min, 12,000 x g, 4 °C (white, gel-like pellet) |
| 75% ethanol wash | 1 mL; spin 5 min, 7,500 x g, 4 °C |
| Air-dry | 5-10 min; the guide says not to vacuum-centrifuge dry |
| Resuspension | 20-50 µL RNase-free water, 0.1 mM EDTA or 0.5% SDS; 55-60 °C for 10-15 min |
Sample handling, quality and yield
- Do not pre-wash cells. The guide says to add TRIzol without washing, because washing can degrade mRNA.
- Process promptly. Samples are homogenised immediately or quick-frozen at -80 °C or in liquid nitrogen. For tissues rich in RNase, such as spleen or pancreas, the guide advises cold TRIzol.
- Stop points. The lysate can be held at 4 °C overnight or at -20 °C for up to a year; RNA in 75% ethanol is stable for at least a year at -20 °C or at least a week at 4 °C; resuspended RNA is stored at -70 °C.
- Small samples. For fewer than 1 x 106 cells or under 10 mg of tissue, the guide suggests an optional 5-10 µg of RNase-free glycogen as a carrier so the pellet is recoverable.
- Quantification. Multiply A260 by the dilution factor and 40 to get µg RNA per mL; an A260/A280 ratio near 2 is considered pure. Fluorescence methods such as Qubit measure intact RNA selectively.
- Typical yield. The guide’s table gives, at A260/280 above 1.8, about 8-15 µg of RNA from 1 x 106 epithelial cells and about 5-7 µg from 1 x 106 fibroblasts (skeletal muscle and brain give about 1-1.5 µg per mg of tissue), illustrating that yield depends heavily on cell type.
Safety and compatibility
TRIzol contains phenol and guanidine isothiocyanate and is used with chloroform. The guide directs users to read the safety data sheet and wear eye protection, protective clothing and gloves, and it requires that any material in contact with the reagents be compatible with phenol, guanidine isothiocyanate and chloroform, which is why polypropylene tubes are specified. Work involving volatile solvents is normally done in a ventilated enclosure such as a fume hood, following your institution’s chemical hygiene plan. Gloves, RNase-free plasticware and an RNase-decontamination product for surfaces keep RNases out of the workflow.
When to use a column kit or a hybrid
The reagent method and silica-column kits solve the same problem differently; see what an RNA extraction kit is for the column approach. The guide itself describes a combined route: phase-separation tubes (Phasemaker, A33248) create a physical barrier between the phases, and the aqueous phase can then be taken through the PureLink RNA Mini Kit spin columns. Isolated RNA is described as suitable for RT-PCR, Northern blot, dot blot, poly(A)+ selection, in vitro translation, RNase protection assays and molecular cloning; downstream amplification is typically run on a thermal cycler.
Common misunderstandings
- “Chloroform is what lyses the cells.” No. Lysis and RNase inhibition come from the phenol and guanidine isothiocyanate in the reagent; chloroform drives phase separation.
- “The upper phase is free of DNA risk.” The DNA sits in the interphase and organic layer. Drawing the aqueous phase too close to the interphase is how it carries over.
- “A dried pellet is a better pellet.” The guide cautions against vacuum drying and over-drying; RNA that does not fully dissolve gives poor purity readings.
- “SDS is always a safe resuspension liquid.” The guide lists 0.5% SDS as an option but not where enzymatic reactions follow.
- “TRIzol is a diagnostic reagent.” The label reads for research use only, not for use in diagnostic procedures.
FAQ
What does TRIzol do in RNA extraction?
It lyses cells and inactivates RNases in one step. With chloroform and centrifugation, it then lets RNA partition into the aqueous phase, away from DNA and protein.
What is the role of chloroform in the TRIzol method?
Chloroform (0.2 mL per 1 mL TRIzol in the guide) causes the homogenate to separate into aqueous, interphase and organic layers. It is not what disrupts the cells.
Is TRIzol a kit?
The reagent is sold in 100 mL and 200 mL packs (100 and 200 reactions per the guide) and is used with separately supplied chloroform, isopropanol, ethanol and RNase-free water. Silica-column products are a different format; the two can be combined as described above.
Where can I find a TRIzol RNA extraction protocol PDF?
The manufacturer’s TRIzol Reagent User Guide (MAN0001271) is the authoritative step-by-step document; this page explains the principles and parameters rather than replacing it.
Does TRIzol extraction include DNase treatment?
The user guide does not describe a DNase step. Whether and how to DNase-treat depends on the downstream assay; see RNA extraction protocol basics.
Sources
- Thermo Fisher Scientific / Invitrogen. TRIzol Reagent User Guide, Pub. No. MAN0001271 Rev. D (Cat. 15596026, 15596018). documents.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf. Read 2026-11-07.
- Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162(1):156-159. PMID 2440339; doi:10.1006/abio.1987.9999. pubmed.ncbi.nlm.nih.gov/2440339. Read 2026-11-07.
