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From Tissue to Transcript: The Protocol for Trizol RNA Extraction

CLYTE research team
Nov 12
6 min read

Trizol reagent is a cornerstone of molecular biology, renowned for its ability to deliver high-quality total RNA from a wide variety of samples. As a ready-to-use, monophasic solution of phenol and guanidine isothiocyanate, its power lies in its aggressive efficiency. During lysis, it simultaneously disrupts cells, denatures proteins, and inactivates the RNases that would otherwise degrade your sample. This makes it the gold-standard method for isolating not just RNA, but also DNA and proteins sequentially from the same sample.

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Ask Sophie about Trizol RNA extraction from tissue!

However, when working with animal tissue, the standard protocol requires critical modifications. The fibrous, complex nature of tissue presents a significant challenge: achieving complete and immediate homogenization to ensure every cell is exposed to the Trizol. This guide synthesizes expert protocols and troubleshooting tips to help you master the art of Trizol extraction from tissue.

The Critical First Step: Tissue Homogenization

You cannot get high-quality RNA if your tissue is not completely homogenized. The goal is to instantly pulverize the tissue in the Trizol to stop all RNase activity. The official Thermo Fisher manual recommends using 1 mL of Trizol Reagent per 50-100 mg of tissue and homogenizing with a mechanical homogenizer.

Based on academic and research protocols, there are three primary methods to achieve this:

The Liquid Nitrogen (Mortar and Pestle) Method: This is the traditional gold standard for tough tissues. The tissue is snap-frozen in liquid nitrogen and then ground into a fine powder using a pre-chilled mortar and pestle. All tools, including the mortar, pestle, and spatula, must be chilled on dry ice beforehand. The fine, frozen tissue powder is then transferred (using a pre-chilled spatula) into a tube containing the Trizol. The cardinal rule: the tissue powder must never, ever thaw before it hits the Trizol.
Mechanical Homogenizer (e.g., TissueLyser): This is a faster, high-throughput method. The NCI's protocol, for example, places a 5 mm stainless steel bead into a 2 mL tube with 0.5 mL of Trizol. The frozen tissue sample (50-70 mg) is added, and the tube is processed in a TissueLyser (e.g., 3 minutes at 25 Hz) until fully homogenized.
Direct Homogenization (Rotor-Stator or Pestle): For some tissues, it's possible to add 1 mL of Trizol directly to the 50-100 mg piece of frozen tissue and immediately homogenize using a rotor-stator homogenizer (like a BioSpec Tissue Tearor) or a microcentrifuge tube pestle until no visible tissue fragments remain.

Pro-Tip for Fatty Tissues: If your sample (e.g., brain or adipose tissue) has a high fat content, the Trizol manual recommends an extra step. After homogenization, centrifuge the lysate for 5 minutes at 12,000 x g before adding chloroform. Transfer the clear, fat-free supernatant to a new tube and proceed.

The Core Trizol RNA Extraction Protocol: A 5-Step Guide

Once your tissue is a uniform liquid homogenate, the standard protocol begins. Note: All centrifugation steps should be performed at 2-8°C or 4°C.

Step 1: Phase Separation

This step uses chloroform to separate the solution into phases.

Incubate the tissue homogenate for 5 minutes at room temperature (15-30°C). This allows nucleoprotein complexes to fully dissociate.
Add 0.2 mL of chloroform per 1 mL of Trizol used.
Cap the tube securely and shake vigorously by hand for 15 seconds. Do not vortex.
Incubate at room temperature for 2-3 minutes.
Centrifuge at ≤12,000 x g for 15 minutes.
You will now see three distinct phases: a lower red phenol-chloroform phase, a milky interphase, and a colorless upper aqueous phase. Your RNA is exclusively in this top aqueous phase.

Step 2: RNA Precipitation

This step uses isopropanol to pull the RNA out of the aqueous phase.

Carefully transfer the upper aqueous phase to a fresh tube. Be extremely careful to avoid drawing up any of the interphase or lower organic layer, as this will contaminate your RNA.
Add 0.5 mL of 100% isopropyl alcohol per 1 mL of initial Trizol used.
(Optional): If you expect very low RNA yield, you can add a carrier like 5-10 µg of RNase-free glycogen or acrylamide at this step.
Gently invert to mix and incubate at room temperature for 10 minutes.
Centrifuge at ≤12,000 x g for 10 minutes.
The RNA precipitate will form a small, gel-like (often invisible or translucent white) pellet on the side and bottom of the tube.

Step 3: RNA Wash

This step washes away salts and other impurities.

Carefully pour off or pipette out the supernatant, discarding it.
Wash the pellet once by adding at least 1 mL of 75% ethanol per 1 mL of initial Trizol used.
Mix by vortexing briefly or inverting the tube.
Centrifuge at ≤7,500 x g for 5 minutes.

Step 4: Solubilization

This is the final step to re-dissolve your pure RNA.

Discard the 75% ethanol supernatant. Use a pipette to remove any remaining drops.
Briefly air-dry the pellet for 5-10 minutes.
CRITICAL: Do not let the pellet dry completely. Over-drying makes the RNA extremely difficult to redissolve and will result in a poor A260/280 ratio (<1.6).
Resuspend the pellet in RNase-free water (the NCI protocol uses 50 µL).
Incubate for 10-15 minutes at 55-60°C to aid dissolution, pipetting up and down a few times.

Step 5: (Optional) DNase Treatment

The Trizol method can carry over trace amounts of genomic DNA (gDNA). If your downstream application (like qPCR) is sensitive to gDNA, a DNase treatment is recommended. This can be done by adding DNase I buffer and DNase I to the final, resuspended RNA and incubating at 37°C. Alternatively, kits like the Trizol Plus RNA Purification Kit combine the powerful Trizol lysis with a silica spin-column cleanup, which includes an easy, on-column DNase treatment step.

Troubleshooting and Pro-Tips from the Bench

Sample Storage: Can you store samples in Trizol? Yes. After homogenization (but before chloroform), samples can be stored at -70°C for at least one month. Many labs do this to batch-process samples. The final RNA pellet (in 75% ethanol, before drying) is stable for at least a year at -20°C.
The -80°C Warning: While storing the initial homogenate in Trizol at -80°C is generally fine, do NOT store the aqueous phase/isopropanol mixture at -80°C. While it might seem like this would increase precipitation, it primarily causes the guanidine salts to crash out with your RNA, leading to severe contamination that inhibits downstream reactions.
"My Trizol turned brown!": This is a common observation, especially with bloody tissues like the liver. In most cases, this is just pigment and does not affect RNA quality. However, if you are using a metal drill or homogenizer tip, a brown color could indicate rust from the Trizol reacting with the metal. This can compromise RNA quality (low RIN) even if Nanodrop ratios appear normal.

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Trizol RNA extraction Frequently Asked Questions (FAQ)

Can TRIzol be used on tissue samples?

Yes, absolutely. TRIzol Reagent is specifically designed and validated for isolating high-quality total RNA from a wide variety of samples, including tissue samples from human, animal, and plant sources. The standard protocol recommends using 50-100 mg of tissue per 1 mL of TRIzol Reagent. It is a standard method used in many labs for processing solid tissues, such as the neuroblastoma tumor samples processed for the NCI's TARGET initiative.

How to extract RNA from cells in TRIzol?

The process for cells is slightly different from tissue as it doesn't require mechanical homogenization.

For cells grown in a monolayer (adherent cells): Lyse the cells directly in the culture dish. First, remove the growth media, then add 1 mL of TRIzol Reagent directly to the dish (for a 3.5-cm dish) and pipet the cell lysate up and down several times to homogenize.
For cells grown in suspension: Harvest the cells by centrifugation to form a pellet. Add 1 mL of TRIzol Reagent to the pellet (per 5-10 x 10^6 cells) and lyse them by repetitive pipetting. The manual specifically notes not to wash the cells before adding the TRIzol, as this can risk mRNA degradation.

After this lysis step, you proceed with the standard Trizol protocol (chloroform addition, phase separation, precipitation, etc.).

How to homogenize tissue in TRIzol?

Complete homogenization is the most critical step for successful RNA extraction from tissue. The goal is to disrupt the tissue immediately upon contact with TRIzol to inactivate RNases. The most common methods are:

Mechanical Homogenizer: Use a rotor-stator homogenizer (like a Tissue Tearor) or a bead-beater (like a TissueLyser). For a TissueLyser, the tissue is often added to a tube containing a 5 mm stainless steel bead and 0.5-1 mL of Trizol, then agitated at high speed (e.g., 3 minutes at 25 Hz) until no solid tissue remains.
Liquid Nitrogen Grinding: This is the traditional gold-standard method. The tissue is snap-frozen in liquid nitrogen, placed in a pre-chilled mortar, and ground into a fine powder using a pestle. This frozen powder is then transferred (using a pre-chilled spatula) directly into the tube containing Trizol. It is essential that the tissue powder does not thaw before being submerged in the Trizol.

Can you extract RNA from fixed tissue?

The standard Trizol protocols provided are optimized for fresh or frozen tissue. The official user manual specifies "Fresh tissues" or tissues stored in a stabilization solution (like RNAlater) as the required starting material. If not processed immediately, samples should be snap-frozen and stored at -80 C. While the standard protocol is not designed for it, specialized protocols do exist. One of the provided sources lists a related publication titled "Preparation of RNA from Paraffin-Embedded Fixed Tissue Using Trizol," which suggests it is possible with a modified procedure.