Molecular Cloning | Protocol 1Print Version

Chapter 7, Protocol 1
Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium ThiocyanateChloroform Extraction
In this single-step technique, cells are homogenized in guanidnium thiocyanate and the RNA is purified from
the lysate by extraction with phenol:chloroform at reduced pH. Many samples can be processed simultaneously
and speedily. The yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7
g/ml starting tissue or 5-10 g/106 cells. IMPORTANT: Prepare all reagents used in this protocol with DEPCtreated H2O.
CAUTION
RECIPE

MATERIALS
Buffers and Solutions
Chloroform:isoamyl alcohol (49:1, v/v)
Ethanol
Formamide (Optional)
Deionized formamide is used for the storage of RNA.
Isopropanol
Liquid nitrogen
Phenol
PBS
Required for cells grown in suspension and monolayers only.
Sodium acetate (2 M, pH 4.0)
Solution D (denaturing solution)
Cells and Tissues
Mammalian cells
Mammalian tissue samples

METHOD
1. Prepare cells or tissue samples for isolation of RNA as appropriate for the material under study. The
table below describes the amounts of Solution D required for each type of sample.
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Amount of Solution D Required to Extract RNA from Cells and Tissues

Amount of Tissue
Amount of
or Cells
Solution D
100 mg of tissue

3 ml

T-75 flask of cells

3 ml

60-mm plate of cells

1 ml

90-mm plate of cells

2 ml

For tissues
a. Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.
b. Transfer approx. 100 mg of the frozen tissue to a mortar containing liquid nitrogen and pulverize the
tissue using a pestle. The tissue can be kept frozen during pulverization by the addition of liquid
nitrogen.
c. Transfer the powdered tissue to a polypropylene snap-cap tube containing 3 ml of Solution D.
d. Homogenize the tissue for 15-30 seconds at room temperature with a polytron homogenizer.
For mammalian cells grown in suspension
a. Harvest the cells by centrifugation at 200-1900g (1000-3000 rpm in a Sorvall RT600 using the
H1000 rotor) for 5-10 minutes at room temperature in a benchtop centrifuge.
b. Remove the medium by aspiration and resuspend the cell pellets in 1-2 ml of sterile ice-cold PBS.
c. Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 2 ml of
Solution D per 106 cells.
d. Homogenize the cells with a polytron homogenizer for 15-30 seconds at room temperature.
For mammalian cells grown in monolayers
a. Remove the medium and rinse the cells once with 5-10 ml of sterile ice-cold PBS.
b. Remove PBS and lyse the cells in 2 ml of Solution D per 90-mm culture dish (1 ml per 60 mm dish).
c. Transfer the cell lysates to a polypropylene snap-cap tube.

2.
3.
4.
5.
6.
7.

8.

d. Homogenize the lysates with a polytron homogenizer for 15-30 seconds at room temperature.
Transfer the homogenate to a fresh polypropylene tube and sequentially add 0.1 ml of 2 M sodium
acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol per milliliter of Solution D.
After addition of each reagent, cap the tube and mix the contents thoroughly by inversion.
Vortex the homogenate vigorously for 10 seconds. Incubate the tube for 15 minutes on ice to permit
complete dissociation of nucleoprotein complexes.
Centrifuge the tube at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4C, and then
transfer the upper aqueous phase containing the extracted RNA to a fresh tube.
Add an equal volume of isopropanol to the extracted RNA. Mix the solution well and allow the RNA to
precipitate for 1 hour or more at -20C.
Collect the precipitated RNA by centrifugation at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 30
minutes at 4C.
Carefully decant the isopropanol and dissolve the RNA pellet in 0.3 ml of Solution D for every 1 ml of
this solution used in Step 1.
IMPORTANT Pellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it until the
pellet has been checked.
Transfer the solution to a microfuge tube, vortex it well, and precipitate the RNA with 1 volume of
isopropanol for 1 hour or more at -20C.

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9. Collect the precipitated RNA by centrifugation at maximum speed for 10 minutes at 4C in a microfuge.
Wash the pellet twice with 75% ethanol, centrifuge again, and remove any remaining ethanol with a
disposable pipette tip. Store the open tube on the bench for a few minutes to allow the ethanol to
evaporate. Do not allow the pellet to dry completely.
10. Add 50-100 l of DEPC-treated H2O. Store the RNA solution at -70C.
Addition of SDS to 0.5% followed by heating to 65C may assist dissolution of the pellet.
11. Estimate the concentration of the RNA by measuring the absorbance at 260 nm of an aliquot of the final
preparation.

RECIPES
Formamide
Deionize the formamide by stirring on a magnetic stirrer with Dowex XG8
mixed bed resin for 1 hour and filtering it twice through Whatman No. 1
paper. Store deionized formamide in small aliquots under nitrogen at 70C.
KCl
Dissolve an appropriate amount of solid KCl in H2O, autoclave for 20
minutes on liquid cycle and store at room temperature. Ideally, this 4 M
solution should be divided into small (approx. 100 l) aliquots in sterile
tubes and each aliquot thereafter used one time.
NaCl
To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust
the volume to 1 liter with H2O. Dispense into aliquots and sterilize by
autoclaving. Store the NaCl solution at room temperature.
PBS
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
2 mM KH2PO4

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(Phosphate-buffered Saline) Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of

Na2HPO4, and 0.24 g of KH2PO4 in 800 ml of distilled H2O. Adjust the pH
to 7.4 with HCl. Add H2O to 1 liter. Dispense the solution into aliquots and
sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on
liquid cycle or by filter sterilization. Store the buffer at room temperature. If
necessary, PBS may be supplemented with 1 mM CaCl2 and 0.5 mM
MgCl2.
Phenol
Most batches of commercial liquified phenol are clear and colorless and
can be used in molecular cloning without redistillation. Occasionally,
batches of liquified phenol are pink or yellow, and these should be rejected
and returned to the manufacturer. Crystalline phenol is not recommended
because it must be redistilled at 160C to remove oxidation products, such
as quinones, that cause the breakdown of phosphodiester bonds or cause
cross-linking of RNA and DNA.Before use, phenol must be equilibrated to a
pH of >7.8 because the DNA partitions into the organic phase at acid pH.
Wear gloves, full face protection, and a lab coat when carrying out this
procedure.
1. Store liquified phenol at -20C. As needed, remove the phenol from the
freezer, allow it to warm to room temperature, and then melt it at 68C. Add
hydroxyquinoline to a final concentration of 0.1%. This compound is an
antioxidant, a partial inhibitor of RNase, and a weak chelator of metal ions.
In addition, its yellow color provides a convenient way to identify the
organic phase.
2. To the melted phenol, add an equal volume of buffer (usually 0.5 M TrisCl [pH 8.0] at room temperature). Stir the mixture on a magnetic stirrer for
15 minutes. Turn off the stirrer, and when the two phases have separated,
aspirate as much as possible of the upper (aqueous) phase using a glass
pipette attached to a vacuum line equipped with appropriate traps.
3. Add an equal volume of 0. 1 M Tris-Cl (pH 8.0) to the phenol. Stir the
mixture on a magnetic stirrer for 15 minutes. Turn off the stirrer and
remove the upper aqueous phase as described in Step 2. Repeat the
extractions until the pH of the phenolic phase is >7.8 (as measured with pH
paper).
4. After the phenol is equilibrated and the final aqueous phase has been
removed, add 0.1 volume of 0.1 M Tris-Cl (pH 8.0) containing 0.2% mercaptoethanol. The phenol solution may be stored in this form under 100
mM Tris-Cl (pH 8.0) in a light-tight bottle at 4C for periods of up to 1
month.
Sodium Acetate

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To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate3H2O in

800 ml of H2O. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH
to 7.0 with dilute acetic acid. Adjust the volume to 1 liter with H2O.
Dispense into aliquots and sterilize by autoclaving.
Solution D
4 M guanidinium thiocyanate

100 gm - Rs 8484/- from Sigma

25 mM sodium citrate2H2O
0.5% (w/v) sodium lauryl sarcosinate 50 gm - Rs 2,988.76 from HiMedia
0.1 M

-mercaptoethanol

To prepare denaturing solution: Dissolve 250 g of guanidinium thiocyanate

in 293 ml of H2O, 17.6 ml of 0.75 M sodium citrate (pH 7.0), and 26.4 ml of
10% (w/v) sodium lauryl sarcosinate. Add a magnetic bar and stir the
solution on a combination heater-stirrer at 65C until all ingredients are
dissolved. Store Solution D at room temperature, and add 0.36 ml of 14.4
M stock -mercaptoethanol per 50 ml of Solution D just before use.
Solution D may be stored for months at room temperature but is sensitive
to light. Note that guanidinium will precipitate at low temperatures.

CAUTIONS
-Mercaptoethanol
-Mercaptoethanol (2-Mercaptoethanol) HOCH2CH2SH may be fatal if inhaled or absorbed through
the skin and is harmful if ingested. High concentrations are extremely destructive to the mucous
membranes, upper respiratory tract, skin, and eyes. -Mercaptoethanol has a very foul odor. Wear
appropriate gloves and safety glasses. Always use in a chemical fume hood.
Chloroform:isoamyl alcohol
Chloroform:isoamyl alcohol, see Chloroform; Isoamyl alcohol
Formamide
Formamide is teratogenic. The vapor is irritating to the eyes, skin, mucous membranes, and upper
respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate
gloves and safety glasses. Always use in a chemical fume hood when working with concentrated
solutions of formamide. Keep working solutions covered as much as possible.
Guanidinium thiocyanate
Guanidinium thiocyanate, see Guanidine thiocyanate
Liquid nitrogen

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Liquid nitrogen can cause severe damage due to extreme temperature. Handle frozen samples with
extreme caution. Do not breathe the vapors. Seepage of liquid nitrogen into frozen vials can result in
an exploding tube upon removal from liquid nitrogen. Use vials with O-rings when possible. Wear cryomitts and a face mask.
Phenol
Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by
inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles, and protective clothing.
Always use in a chemical fume hood. Rinse any areas of skin that come in contact with phenol with a
large volume of water and wash with soap and water; do not use ethanol!

REFERENCES
1. Chomczynski P. and Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162:156-159.

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