USER GUIDETRIzol™ Reagent
Catalog Numbers 15596026 and 15596018
Doc. Part No. 15596026.PPSPub. No. MAN0001271 Rev. C.0

WARNING! Read the Safety Data Sheets (SDSs) andfollow the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves. Safety Data Sheets (SDSs) areavailable from thermofisher.com/support.

Item

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Invitrogen TRIzol Reagent is a ready-to-use reagent, designed toisolate high quality total RNA (as well as DNA and proteins) from celland tissue samples of human, animal, plant, yeast, or bacterial origin,™
within one hour. TRIzol Reagent is a monophasic solution of phenol,
guanidine isothiocyanate, and other proprietary components whichfacilitate the isolation of a variety of RNA species of large or small™
molecular size. TRIzol Reagent maintains the integrity of the RNA
due to highly effective inhibition of RNase activity while disrupting
cells and dissolving cell components during sample homogenization.
™
TRIzol Reagent allows for simultaneous processing of a large
number of samples, and is an improvement to the single-step RNA
isolation method.
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TRIzol Reagent allows users to perform sequential precipitation ofRNA, DNA, and proteins from a single sample. After homogenizing™
the sample with TRIzol Reagent, chloroform is added, and thehomogenate is allowed to separate into a clear upper aqueouslayer (containing RNA), an interphase, and a red lower organiclayer (containing the DNA and proteins). RNA is precipitated fromthe aqueous layer with isopropanol. DNA is precipitated from theinterphase/organic layer with ethanol. Protein is precipitated fromthe phenol-ethanol supernatant by isopropanol precipitation. The
precipitated RNA, DNA, or protein is washed to remove impurities,
and then resuspended for use in downstream applications.
• Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot
Blot hybridization, poly(A)+ selection, in vitro translation, RNase
protection assay, and molecular cloning.
• Isolated DNA can be used in PCR, Restriction Enzyme digestion,and Southern Blots.
• Isolated protein can be used for Western Blots, recovery of someenzymatic activity, and some immunoprecipitation.
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For DNA isolation, see the TRIzol Reagent (DNA isolation) User Guide(Pub. No. MAN0016385).
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TRIzol Reagent can also be used with Phasemaker Tubes (Cat. No.
™
A33248) to isolate RNA. Phasemaker Tubes creates a solid barrier™
between the organic and aqueous phases of the TRIzol Reagent
following sample homogenization which makes separation of phases™
™
easier. See the TRIzol Reagent and Phasemaker Tubes Complete
System User Guide (Pub. No. MAN0016163) for the full protocol.
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TRIzol Reagent can also be used with the PureLink RNA Mini Kit(Cat. No. 12183018A ) which uses spin columns instead of ethanolprecipitation to purify the RNA. For additional information, see the™
PureLink RNA Mini Kit User Guide (Pub. No. MAN0000406).

Contents

Cat. No. 15596026(100 reactions)Cat. No. 15596018(200 reactions)

Storage

TRIzol Reagent

100 mL

200 mL

2–25°C

SourceEquipment
Centrifuge and rotor capable of reaching
12,000 × g and 4°C
Tubes

MLS

Polypropylene microcentrifuge tubes
Reagents

MLS

Chloroform

MLS

Table 2 Materials required for RNA isolation
SourceItem
Equipment
Water bath or heat block at 55–60°C
Reagents

MLS

Isopropanol
Ethanol, 75%
RNase-free water or 0.5% SDS(Optional) RNase-free glycogen or 0.1 mM EDTA

MLSMLS
MLS
MLS

Table 3 Materials required for protein isolation
SourceItem
Equipment(Optional) Dialysis membranes
Reagents

MLS

Isopropanol
Ethanol, 100%0.3 M Guanidine hydrochloride in 95% ethanol
1% SDS

MLS
MLS
MLS
MLS

Input sample requirements
IMPORTANT! Perform RNA isolation immediately after sample
collection or quick-freeze samples immediately after collection andstore at –80°C or in liquid nitrogen until RNA isolation.
Sample typeTissues[1]Cells grown in monolayer

Cells grown in suspension

Contents and storage™

Unless otherwise indicated, all materials are available throughthermofisher.com. "MLS" indicates that the material is available fromfisherscientific.com or another major laboratory supplier.
Table 1 Materials required for all isolations

Product description™

Required materials not supplied

Starting material per 1 ml of TRIzol
reagent
50–100 mg of tissue
1 × 105–1 × 107 cells grown inmonolayer in a 3.5–cm culture dish(10 cm2)
5–10 × 106 cells from animal, plant,or yeast origin or 1 × 107 cells ofbacterial origin[1] Fresh tissues or tissues stored in RNAlater™ Stabilization Solution (Cat.

No. AM7020).

For Research Use Only. Not for use in diagnostic procedures.

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b. Add 1 mL of TRIzol Reagent per 0.25 mL of sample (5–10 × 106 cells from animal, plant, or yeast origin or 1 ×107
cells of bacterial origin) to the pellet.

Procedural guidelines• Perform all steps at room temperature (20–25°C) unless otherwise
noted.
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• Use cold TRIzol Reagent if the starting material contains highlevels of RNase, such as spleen or pancreas samples.
• Use disposable, individually wrapped, sterile plasticware andsterile, disposable RNA-free pipettes, pipette tips, and tubes.
• Wear disposable gloves while handling reagents and RNA
samples to prevent RNase contamination from the surface ofthe skin; change gloves frequently, particularly as the protocol
progresses from crude extracts to more purified materials.
• Always use proper microbiological aseptic techniques whenworking with RNA.
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• Use RNaseZap RNase Decontamination Solution (Cat.
no. AM9780) to remove RNase contamination from work surfaces
and non-disposable items such as centrifuges and pipettes usedduring purification.
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• Ensure that all materials that come into contact with TRIzol
Reagent are compatible with phenol, guanidine isothiocyanate,and chloroform.

Lyse samples and separate phases™

1. Lyse and homogenize samples in TRIzol Reagent according to
your starting material.
• Tissues:™

Add 1 mL of TRIzol Reagent per 50–100 mg of tissue to thesample and homogenize using a homogenizer.
• Cell grown in monolayer:
a. Remove growth media.
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b. Add 1 mL of TRIzol Reagent per 1 × 105—1 × 107 cells
directly to the 3.5–cm culture dish to lyse the cells.
c. Pipet the lysate up and down several times to homogenize.
• Cells grown in suspension:a. Collect the cells by centrifugation and discard thesupernatant.

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Note: Do not wash cells before addition of TRIzol Reagent
to avoid mRNA degradation.
c. Pipet the lysate up and down several times to homogenize.
Note: The sample volume should not exceed 10% of the volume™
of TRIzol Reagent used for lysis.

2.
3.
4.
5.
6.

STOPPING POINT Samples can be stored at 4°C overnight or at–20°C for up to a year.
(Optional) If samples have a high fat content, centrifuge the lysatefor 5 minutes at 12,000 × g at 4–10°C, then transfer the clear
supernatant to a new tube.
Incubate for 5 minutes to allow complete dissociation of thenucleoproteins complex.
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Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used forlysis, securely cap the tube, then thoroughly mix by shaking.
Incubate for 2–3 minutes.
Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.

The mixture separates into a lower phenol-chloroform, aninterphase, and a colorless upper aqueous phase.
7. Transfer the aqueous phase containing the RNA to a new tube byangling the tube at 45° and pipetting the solution out.
IMPORTANT! Avoid transferring any of the interphase or organic
layer into the pipette when removing the aqueous phase.
Proceed directly to “Isolate RNA” on page 2.
To isolate DNA or protein, save the interphase and organic™
phase. See the TRIzol Reagent (DNA isolation) User Guide(Pub. No. MAN0016385) or see “Isolate proteins” on page 3
for detailed procedures. The organic phase can be stored at 4°C
overnight.

Isolate RNA

1

Precipitate the RNA

1.1. (Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 µg of RNase-freeglycogen as a carrier to the aqueous phase.
Note: The glycogen is co-precipitated with the RNA, but does not interfere with subsequent
applications.
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1.2. Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol Reagent used for lysis.
1.3. Incubate for 10 minutes at 4°C.
1.4. Centrifuge for 10 minutes at 12,000 × g at 4°C.
Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
1.5. Discard the supernatant with a micropipettor.

2

Wash the RNA™

2.1. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol Reagent used for lysis.
Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.
2.2. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
2.3. Discard the supernatant with a micropipettor.
2.4. Vacuum or air dry the RNA pellet for 5–10 minutes.
IMPORTANT! Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure totalsolubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.

3Solubilize the RNA

3.1. Resuspend the pellet in 20–50 µL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution bypipetting up and down.
IMPORTANT! Do not dissolve the RNA in 0.5% SDS if the RNA is to be used in subsequent enzymatic
reactions.
3.2. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
Proceed to downstream applications, or store the RNA at –70°C.

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TRIzol Reagent User Guide

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Determine the RNA yield

Determine the RNA yield using one of the following methods.
MethodAbsorbanceAbsorbance at 260 nm provides total
nucleic acid content, while absorbance at
280 nm determines sample purity. Since
free nucleotides, RNA, ssDNA, and dsDNA
absorb at 260 nm, they all contribute to thetotal absorbance of the sample.

Fluorescence
Fluorescence selectively measures intact
RNA, but does not measure protein orother contaminants present in the sample

Procedure
1. Dilute sample in RNase-free water, then measure absorbance at260 nm and 280 nm.
2. Calculate the RNA concentration using the formula A260 × dilution
× 40 = µg RNA/mL.
3. Calculate the A260/A280 ratio.
A ratio of ~2 is considered pure.
RNA samples can be quantified by absorbance without prior dilution™
using the NanoDrop Spectrophotometer. Refer to the instruments
instructions for more information.
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• Quantify RNA yield using the appropriate Qubit or Quant-iT RNA
Assay Kit (Cat. Nos. Q32852, Q10210, Q33140, or Q10213).
Refer to the kits instructions for more information.

Table 4 Typical RNA (A260/280 of >1.8) yields from various starting materials
Starting material
Epithelial cells
New tobacco leaf
Fibroblasts
Skeletal muscles and brain
Placenta
LiverKidney

Quantity

RNA yield

1 × 106 cells—1 × 106 cells
1 mg
1 mg
1 mg
1 mg

8–15 µg
73 µg
5–7 µg
1–1.5 µg
1–4 µg
6–10 µg
3–4 µgIsolate proteins

Isolate the proteins from the organic phase saved from “Isolate RNA” on page 2 using either “Precipitate the proteins” on page 3 or “Dialyse theproteins” on page 4.

1

Precipitate the proteins

Remove any remaining aqueous phase overlying the interphase.
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Add 0.3 mL of 100% ethanol per 1 mL of TRIzol Reagent used for lysis.
Cap the tube, mix by inverting the tube several times.
Incubate for 2–3 minutes.
Centrifuge for 5 minutes at 2000 × g at 4°C to pellet the DNA.
Transfer the phenol-ethanol supernatant to a new tube.
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Add 1.5 mL of isopropanol to the phenol-ethanol supernatant per 1 mL of TRIzol Reagent used forlysis.
1.8. Incubate for 10 minutes.
1.9. Centrifuge for 10 minutes at 12,000 × g at 4°C to pellet the proteins.
1.10. Discard the supernatant with a micropipettor.

2

Wash the proteins

2.1. Prepare a wash solution consisting of 0.3 M guanidine hydrochloride in 95% ethanol.
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2.2. Resuspend the pellet in 2 mL of wash solution per 1 mL of TRIzol Reagent used for lysis.
2.3. Incubate for 20 minutes.

1.1.
1.2.
1.3.
1.4.
1.5.
1.6.
1.7.

2.4.
2.5.
2.6.
2.7.
2.8.
2.9.
2.10.
2.11.

Note: The proteins can be stored in wash solution for at least 1 month at 4°C or for at least 1 year at–20°C.
Centrifuge for 5 minutes at 7500 × g at 4°C.
Discard the supernatant with a micropipettor.
Repeat step 2.2–step 2.5 twice.
Add 2 mL of 100% ethanol, then mix by vortexing briefly.
Incubate for 20 minutes.
Centrifuge for 5 minutes at 7500 × g at 4°C.
Discard the supernatant with a micropipettor.
Air dry the protein pellet for 5–10 minutes.
IMPORTANT! Do not dry the pellet by vacuum centrifuge.

3

Solubilize the proteins

3.1. Resuspend the pellet in 200 µL of 1% SDS by pipetting up and down.
Note: To ensure complete resuspension of the pellet, we recommend that you incubate the sample at50°C in a water bath or heat block.
3.2. Centrifuge for 10 minutes at 10,000 × g at 4°C to remove insoluble materials.
3.3. Transfer the supernatant to a new tube.
Measure protein concentration by Bradford assay (SDS concentration mush be <0.1%), then proceed
directly to downstream applications, or store the sample at –20°C.

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TRIzol Reagent User Guide3Dialyse the proteins
1. Remove any remaining aqueous phase overlying the interphase.
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2. Add 0.3 mL of 100% ethanol per 1 mL of TRIzol Reagent usedfor lysis.
3. Cap the tube, mix by inverting the tube several times.
4. Incubate for 2–3 minutes.
5. Centrifuge for 5 minutes at 2000 × g at 4°C to pellet the DNA.
6. Load the phenol-ethanol supernatant into the dialysis membrane.
Note: The phenol-ethanol solution can dissolve some types ofdialysis membranes (cellulose ester, for example). Test dialysis
tubing with the membrane to assess compatibility before starting.
7. Dialyze the sample against 3 changes of 0.1% SDS at 4°C. Make
the first change of solution after 16 hours, the second change 4hours later (at 20 hours), and the final change 2 hours later (at 22hours).

Proceed directly to downstream applications, or store the sample at–20°C.

Troubleshooting
Troubleshooting and FAQs

Visit our online FAQ database for tips and tricks for conducting yourexperiment, troubleshooting information, and FAQs. The online FAQ
database is frequently updated to ensure accurate and thoroughcontent.
• For troubleshooting information and FAQs for this product:
https://www.thermofisher.com/trizolfaqs• To browse the database and search using keywords:
thermofisher.com/faqs

Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant theirproducts as set forth in the Life Technologies' General Terms andConditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, pleasecontact Life Technologies at www.thermofisher.com/support.

Note: A SDS concentration of at least 0.1% is required toresolubilize the proteins from the pellet. If desired, the SDS canbe diluted after solubilization.
8. Centrifuge the dialysate for 10 minutes at 10,000 × g at 4°C.
9. Transfer the supernatant containing the proteins to a new tube.
10. (Optional) Solubilize the pellet by adding 100 µL of 1% SDS and100 µL of 8 M urea.

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Revision history: Pub. No. MAN0001271
Revision

Date

C.0

14 February 2023

Storage temperature in contents and storage table was changed and typographical errors were corrected.

Description

B.0

28 January 2020

• Added note to ensure that materials that come into contact with the Trizol Reagent are compatible.
• Updated step 4 to include shaking the sample.

A.0

9 November 2016

Added references to Phasemaker Tubes .

–13 December 2012

Baseline for revision.

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The information in this guide is subject to change without notice.
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Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
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14 February 2023