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Protocol

Purification of RNA Using TRIzol (TRI Reagent)

Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen

INTRODUCTION
TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing
RNA. This method is particularly advantageous in situations where cells or tissues are enriched for
endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol
(or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously
solubilizes biological material and denatures protein. After solubilization, the addition of chloroform
causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein
is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous
phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name
TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-
associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets
can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction
is practical.

RELATED INFORMATION
TRIzol extraction is a straightforward way to prepare RNA from cells or tissues. Although TRIzol
removes large DNA molecules efficiently, it does not remove plasmid DNA or DNA fragments efficiently,
which can be problematic for subsequent polymerase chain reaction (PCR) applications. However,
when used appropriately, the RNA yield is quantitative. An alternative method for Purification of RNA
by SDS Solubilization and Phenol Extraction (Rio et al. 2010a) is available, as are procedures for
Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells (Rio et al. 2010b), and
Ethanol Precipitation of RNA and the Use of Carriers (Rio et al. 2010c). Further information is also
available on Determining the Yield and Quality of Purified RNA (Rio et al. 2010d).

MATERIALS
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and
recipes for reagents marked with <R>.
Perform all procedures under RNase-free conditions using RNase-free glassware and other equipment, and
prepare all reagents with RNase-free H2O.

Reagents
Cells or tissues for extraction
<!>Chloroform
Ethanol
Glycogen (or GlycoBlue [Ambion]) (optional; see Step 6)
H2O, RNase-free
<!>Isopropanol
<!>Phenol

Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel

Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold
Spring Harbor, NY, USA, 2010.
Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5439 www.cshprotocols.org

© 2010 Cold Spring Harbor Laboratory Press 1 Vol. 2010, Issue 6, June
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<R>SDS solubilization buffer (1X)

Sodium acetate (NaOAc)
<!>TRIzol (Invitrogen) or TRI Reagent (Molecular Research Center, Inc.)
<!>TRIzol LS (for liquid samples or yeast and bacteria; see Step 1.v)

Equipment
Centrifuge (for large-scale isolations)
<!>Dry ice
<!> Alternatively, liquid nitrogen can be used with a precooled mortar and pestle to prepare the tissue. A
mechanical homogenizer is a less desirable alternative (see Step 1.i).
Freezers preset to -20°C and -80°C
Hood (for chloroform and phenol steps)
Microcentrifuge (for small-scale isolations)
Pasteur pipettes, glass, 5- or 10-mL (pulled for Step 9)
Tubes, microcentrifuge, 1.5-mL (for small-scale isolations)
Tubes, polypropylene, 12-mL, equipped with caps (Sarstedt) (for large-scale isolations)
Vortex mixer

METHOD
Sample Preparation
Completely solubilized samples can be kept at room temperature for, at most, a few hours. Alternatively, freeze for later
use at -80°C. The RNA is stable indefinitely at -80°C.

1. Prepare the samples as required:

For tissues
i. Snap-freeze the tissue to be extracted. Powder on dry ice. Alternatively, using a precooled
mortar and pestle, grind the frozen tissue under liquid nitrogen.
A less desirable alternative is to use a mechanical homogenizer.

ii. Add 1 mL of TRIzol per 50-100 mg of tissue. For larger amounts, scale up accordingly.
Never let the sample volume exceed 10% of the TRIzol volume. It is essential to dissolve the tissues as
quickly as possible in TRIzol.

For cells
iii. Add 1 mL of TRIzol per 1 × 106 tissue culture cells. For larger amounts, scale up accordingly.
Never let the sample volume exceed 10% of the TRIzol volume. It is essential to dissolve the cells as quickly
as possible in TRIzol.

iv. Collect the cells by centrifugation. (For details on cell preparation, see Preparation of
Cytoplasmic and Nuclear RNA from Tissue Culture Cells [Rio et al. 2010b]). Resuspend
the cell pellets in TRIzol by pipetting with a Pasteur pipette, inverting the tubes, or vortexing.
For liquid samples
v. Add 3 volumes of TRIzol LS to 1 volume of liquid sample.

Extraction

2. Solubilize the samples (from Steps 1.ii, 1.iv, or 1.v) for 5 min at room temperature.

3. Add 0.2 mL of chloroform per mL of TRIzol. Mix vigorously by hand or vortex for 15 sec. Allow
the mixture to stand for 2-3 min at room temperature.
Adding chloroform promotes phase separation. It is important that the chloroform does not contain any
additives such as isoamyl alcohol, as used in Purification of RNA by SDS Solubilization and Phenol Extraction
(Rio et al. 2010a).

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4. Centrifuge at maximum speed in a microcentrifuge or at 10,000g in a centrifuge for 10 min at

room temperature.
This should produce two phases: an upper clear one containing RNA and a lower red one that contains protein.
DNA is at the interface.

5. Transfer the upper clear phase to a fresh tube, taking care not to disturb the interface.

6. (Optional) If the expected RNA concentration is 10 µg/mL, add a carrier (e.g., glycogen or
GlycoBlue) (see Ethanol Precipitation of RNA and the Use of Carriers [Rio et al. 2010c]) to the
upper phase before proceeding to Step 7.
7. Add 0.5 mL of isopropanol per each milliliter of the clear phase. Mix vigorously by rapid shaking
or vortexing. Let stand for 10 min.
The isopropanol will precipitate the RNA.

8. Collect the precipitated RNA by centrifugation at maximum speed in a microcentrifuge or at

10,000g in a centrifuge for 10 min at 4°C.
The use of GlycoBlue as a carrier greatly aids in visualizing the pellet.

9. Carefully decant the supernatant. Remove any remaining liquid with a pulled Pasteur pipette.
The white (or blue, if GlycoBlue has been used) RNA pellet will appear as a triangle extending from the bottom
of the tube slightly upward.

10. Immediately resuspend the pellet (without drying) in 1X SDS solubilization buffer.
Although the suppliers of TRI Reagent and TRIzol recommend washing the isopropanol pellet by vortexing with
75% ethanol, this step is not advised. Immediate resuspension followed by phenol extraction gives much cleaner
RNA. Because of the extremely high salt concentration present in TRIzol, the pellet can be difficult to resuspend.
Resuspension can take several minutes, but if GlycoBlue has been used, it is easy to visualize this process.

11. After resuspension, add NaOAc to 3 M.

12. Re-extract the solution with phenol (see Purification of RNA by SDS Solubilization and Phenol
Extraction [Rio et al. 2010a]).
13. Precipitate with ethanol (see Ethanol Precipitation of RNA and the Use of Carriers [Rio
et al. 2010c]).
To assess the quantity and quality of the RNA, see Determining the Yield and Quality of Purified RNA (Rio et
al. 2010d).
See Troubleshooting.

14. Store the purified RNA in ethanol at -20°C.

Alternatively, recover the RNA by centrifugation, resuspend in RNase-free H2O, and store at -20°C (or at -80°C
for long-term storage).

TROUBLESHOOTING
Problem: RNA yield is low.
[Step 13]
Solution: Unexpectedly low RNA yield can occur when the initial solubilization step is not effective;
e.g., it is too short or not enough TRIzol has been used. Make sure that the solution is clear and of
uniform viscosity.

REFERENCES
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. 2010a. Purification of RNA
by SDS solubilization and phenol extraction. Cold Spring Harb
Protoc (this issue). doi: 10.1101/pdb.prot5438.
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. 2010b. Preparation of
cytoplasmic and nuclear RNA from tissue culture cells. Cold Spring
Harb Protoc (this issue). doi: 10.1101/pdb.prot5441.
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. 2010c. Ethanol precipitation
of RNA and the use of carriers. Cold Spring Harb Protoc (this issue).
doi: 10.1101/pdb.prot5440.
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. 2010d. Determining the
yield and quality of purified RNA. Cold Spring Harb Protoc (this
issue). doi: 10.1101/pdb.top82.

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