WEILL CORNELL MEDICAL COLLEGE P. ZUMBO
LABORATORY OF CHRISTOPHER E. MASON, PH.D.
DEPARTMENT OF PHYSIOLOGY & BIOPHYSICS
1
Phenol-chloroform Extraction
Introduction
A phenol-chloroform extraction is a liquid-liquid extraction. A liquid-liquid extraction is a
method that separates mixtures of molecules based on the differential solubilities of the
individual molecules in two different immiscible liquids (28). Liquid-liquid extractions are
widely used to isolate RNA, DNA, or proteins1.
Brief History
Volkin & Carter reported the first use of guanidinium chloride in the isolation of RNA in 1951
(30). In 1953, Grassmann & Defner described the efficacy of phenol at extracting proteins
from aqueous solution (16). Utilizing this find, Kirby demonstrated the use of phenol to
separate nucleic acids from proteins in 1956 (18). Cox and others renewed interest in the use
of guanidinium chloride in the isolation of RNA from ribonucleoproteins in the 1960s
(11,12,13). From then on, guanidinium extractions were the method of choice for RNA
purification, replacing phenol extraction. The use of guanidinium thiocyanate instead of
guanidinium chloride was first briefly mentioned by Ullrich et al. in 1977 (29), and later
successfully employed by Chirgwin et al. in 1979 (8). Chirgwin et al. used guanidinium
thiocyanate to isolate undegraded RNA from ribonuclease-rich tissues like pancreas. A
combination of guanidinium thiocyanate and hot phenol for RNA isolation was reported by
Feramisco et al. in 1981 (14). In 1987, Chomczynski & Sacchi combined guanidinium
thiocyanate with phenol-chloroform extraction under acidic conditions (9). Since its
inception, the Chomczynski & Sacchi method has been the method of choice to isolate RNA
from cultured cells and most animal tissues (10).
Extraction of Nucleic Acids
The extraction of nucleic acids involves adding an equal volume of phenol-chloroform to an
aqueous solution of lysed cells or homogenized tissue, mixing the two phases, and allowing
the phases to separate by centrifugation (Figure 1). Centrifugation of the mixture yields two
phases: the lower organic phase and the upper aqueous phase.
Chloroform mixed with phenol is more efficient at denaturing proteins than either reagent is
alone. The phenol-chloroform combination reduces the partitioning of poly(A)+ mRNA into
the
11 Abbreviations used: RNA, ribonucleic acid; DNA, deoxyribonucleic acid; poly(A)+, polyadenylated;
phenol:CHCl3, phenol-chloroform; mRNA, messenger ribonucleic acid; RNase(s), ribonuclease(s).
2
the organic phase and reduces the formation of insoluble RNAprotein
complexes at the interphase (24). Moreover, phenol retains
about 10-15% of the aqueous phase, which results in a similar loss of
RNA; chloroform prevents this retention of water and thus improves
RNA yield (22). Typical mixtures of phenol to chloroform are 1:1 and
5:1 (v/v). At acidic pH, a 5:1 ratio results in the absence of DNA from
the upper aqueous phase; whereas a 1:1 ratio, while providing
maximal recovery of all RNAs, will maintain some DNA present in the
upper aqueous phase (17). Isoamyl alcohol is sometimes added to
prevent foaming (typically in a ratio of 24 parts chloroform to 1 part
isoamyl alcohol). Guanidinium salts are used to reduce the effect of
nucleases.
Purified phenol has a density of 1.07 g/cm3 and therefore forms the
lower phase when mixed with water (1.00 g/cm3) (21). Chloroform
ensures phase separation of the two liquids because chloroform is
miscible with phenol and it has a higher density (1.47 g/cm3) than
phenol (21); it forces a sharper separation of the organic and
aqueous phases thereby assisting in the removal of the aqueous
phase with minimal cross contamination from the organic phase.
In general, a solute dissolves best in a solvent that is most similar in
chemical structure to itself. The overall solvation capacity of a
solvent depends primarily on its polarity (20). For example, a very
polar solute such as urea is very soluble in highly polar water, less
soluble in fairly polar methanol, and almost insoluble in non-polar
solvents such as chloroform and ether (21). Nucleic acids are polar
because of their negatively charged phosphate backbone, and
therefore nucleic acids are soluble in the upper aqueous phase
instead of the lower organic phase (water is more polar than phenol)
(20). Conversely, proteins contain varying proportions of charged
and uncharged domains, producing hydrophobic and hydrophilic
regions (3). In the presence of phenol, the hydrophobic cores
interact with phenol, causing precipitation of proteins and polymers
(including carbohydrates) to collect at the interface between the
two phases (often as a white flocculent) or for lipids to dissolve in the
lower organic phase (3).
The pH of phenol determines the partitioning of DNA and RNA between the organic phase
and the aqueous phase (6,23). At neutral or slightly alkaline pH (pH 7-8), the phosphate
diesters in nucleic acids are negatively charged, and thus DNA and RNA both partition into
the aqueous phase. DNA is removed from the aqueous layer with increasing efficiency as
the pH is lowered with a maximum efficiency at pH 4.8. At this acidic pH, most proteins and
small DNA fragments (<10 kb) fractionate into the organic phase and large DNA fragments
and some proteins remain at the interphase between the organic and aqueous phases
FIGURE 1. The procedure
of phenol–CHCl3
extraction. Afterward,
the product is cleaned
by alcohol precipitation
or column purification.
3
(6,9,25). Acidic phenol retains RNA in the aqueous phase, but moves DNA into the phenol
phase, because the phosphate groups on the DNA are more easily neutralized than those in
RNA (i.e., DNA is less acidic/has a greater pKa than RNA) (Figure 2) (5,26). An acid pH also
minimizes RNase activity (7).
FIGURE 2. Acid phenol specifically leaves RNA in the aqueous phase. As the pH decreases,
the concentration of protons increases. DNA carries a negative charge because of the
phosphate groups in its sugar-phosphate backbone, which are neutralized in acid by
protonation. In this case, DNA dissolves in the organic phase (like dissolves like). RNA, on the
other hand, is not neutralized in acid because, even though it also has a negative charge, it
has exposed nitrogenous bases (it is single-stranded), which can form hydrogen bonds with
water, keeping it in the aqueous phase. (3)
4
Protocol for Isolation of RNA from Animal Cells or Tissues
Materials
Reagents
• TRIzol reagent
• Chloroform
Miscellanea
You will need access to a fume hood,
vortexer, micropipettes, chilled
microcentrifuge, pellet pestle
homogenizer, and all standard equipment
of a biochemistry-molecular biology
laboratory. ▲ CRITICAL Maximum care
should be taken to not contaminate
samples with RNases. For this reason, use
pipettes reserved for RNA work to prevent
cross-contamination with RNases from
shared equipment. Always wear
disposable gloves, as skin cells as well as
bacteria and molds can contaminate
samples and can be sources of RNases.
Use either disposable, sterile plasticware or
nondisposable glassware or plasticware
that is RNase-free. For this, glassware
should be cleaned with a detergent,
thoroughly rinsed, and oven baked at 240
°C for at least 4 h. Since autoclaving alone
will not fully inactivate many RNases,
glassware can be treated with
diethylpyrocarbonate (DEPC). Fill
glassware with 0.1% DEPC (0.1% in water),
incubate for 12 h at 37 °C, and then
autoclave or heat to 100 °C for 15 min to
eliminate residual DEPC. Plasticware can
be soaked for 10 min in 0.5 M NaOH, 1 mM
EDTA followed by RNase-free water.
Chloroform-resistant plasticware can be
rinsed with chloroform to inactivate
RNases. (15) !	
  CAUTION TRIzol reagent and
chloroform are both toxic and therefore
should be handled under a fume hood
(1,2). .
Setup
Before beginning, clean the workstation
(pipettor shaft, benchtop) with a surface
decontamination solution that destroys
RNases (such as RNase-zap); and chill the
microcentrifuge to 4 ºC. .
Procedure
Homogenization
1| Use option A for tissue or option B for cultured cells.
▲CRITICAL Procedure is done at room temperature (15-25 ºC) unless otherwise indicated.
(A) Fresh tissue is preferable for optimal RNA isolation. Alternatively, tissue should be
submerged in RNAlater stabilization reagent immediately after dissection and stored at –80
°C. Add 1 ml TRIzol reagent per 100 mg fresh tissue, mince on ice using sterile scalpels, and
homogenize with a sterile pellet pestle probe. Frozen tissue should be thawed and removed
from RNAlater stabilization reagent prior to the addition of TRIzol.
(B) Cell cultures should be processed immediately after removal from the incubator.
Either centrifuge cells grown in suspension at 300 x g for 5 min at RT (15-25 ºC) and discard
supernatant or remove the culture medium from cells grown in monolayer. In both cases, it is
not necessary to wash the cells with saline. Add 1 ml TRIzol reagent per 1 × 107 cells to cell
pellets or directly to the culture dish or flask for cells grown in monolayer. Resuspend the
lysate with a sterile, disposable 1-ml pipette tip.
5
Extraction
2| Transfer the tissue or cell lysate to a 2-ml siliconized low-retention tube.
3| Pass sample through sterile, disposable 21 g needle 10 times. In doing so, you will
fragment high-molecular weight cellular components (DNA), thus minimizing their
presence in the aqueous phase.
4| Let homogenate sit at RT (15-25 ºC) for 5 min for complete dissociation of
nucleoprotein complexes.
■ PAUSE POINT You can stop at this point, store your samples at –20 °C, and complete the
procedure later.
5| Add 200 ul chloroform. Vortex vigorously for 15 s.
▲CRITICAL Thorough mixing is important for subsequent phase separation.
▲CRITICAL When mixing and shaking, make sure that the caps are tightly closed!
6| Let homogenate sit at RT (15-25 ºC) for 3 min.
7| Centrifuge at 12,000 x g for 15 min at 4°C. After centrifugation, heat the centrifuge to
room temperature (15–25°C).
8| Transfer upper aqueous phase (600 ul) to new 1.5 ml RNase-free tube.
▲CRITICAL Volume of lysate may be less than 600 ul due to loss during homogenization and
centrifugation.
9| Proceed to either silica-gel membrane purification or alcohol precipitation.
● TIMING
Cell or tissue homogenization and RNA extraction: less than 1 h.
? TROUBLESHOOTING
Troubleshooting is discussed in Table 1.
TABLE 1 | Troubleshooting table.
PROBLEM POSSIBLE REASON SOLUTION
Incomplete phase separation a) No chloroform added
or chloroform not pure
Make sure to add chloroform
that does not contain additives.
b) Homogenate not
sufficiently mixed before
centrifugation
c) Organic solvents in
samples used for RNA
purification
After addition of chloroform, the
homogenate must be vigorously
shaken. If the phases are not
well separated, shake the tube
vigorously for at least 15 s, and
repeat the incubation and
centrifugation in steps 6 and 7.
Ensure starting sample does not
contain organic solvents (e.g.,
ethanol, DMSO), strong buffers,
or alkaline reagents. These can
interfere with phase separation.
6
References
1. Agency for Toxic Substances and Disease Registry. Toxicological Profile for Chloroform.
Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service; 1997.
2. Agency for Toxic Substances and Disease Registry. Toxicological Profile for Phenol.
Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service; 2008
3. Avison M. Measuring Gene Expression. 1st edition. New York: Taylor & Francis Group; 2007.
4. Bowtell D and Sambrook J. DNA Microarrays: A Molecular Cloning Manual. 1st edition.
New York: Cold Spring Harbor Laboratory Press; 2002.
5. Bradley J, Bradley J, Johnson D and Rubenstein D. Lecture Notes on Molecular Medicine.
2nd Edition. Oxford: Blackwell; 2001.
6. Brawerman G, Mendecki J and Lee SY. "A Procedure for the Isolation of Mammalian
Messenger Ribonucleic Acid". Biochemistry 11 (4); 1972.
7. Brown DD. in Methods in Developmental Biology. Wilt FH and Wessells NK, Eds. New York:
Thomas Crowell Co; 1967.
8. Chirgwin JM, Przybyla AE, MacDonald RJ, and Rutter WJ. "Isolation of biologically active
ribonucleic acid from sources enriched in ribonuclease”. Biochemistry, 18:5294-5299; 1979.
9. Chomczynski P and Sacchi N. "Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction". Anal. Biochem. 162: 156–159; 1987.
10.Chomczynski P and Sacchi N. "The single-step method of RNA isolation by acid
guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on".
Nature Protocols 1(2): 581-585; 2006.
11.Cox RA and Arnstein HRV. Biochem. “The isolation, characterization and acid–base
properties of ribonucleic acid from rabbit-reticulocyte ribosomes”. J. 89, 574; 1963.
12.Cox RA. "The use of guanidinium chloride in the isolation of nucleic acids" in Methods in
Enzymology (Grossman L & Moldave K., Eds.), 12-B: 120-129, Orlando FL: Academic Press;
1968
13.Cox RA. “Ribonucleic acid from ribonucleoprotein particles”. Biochem. Prep. 11: 104-109;
1966.
14.Feramisco JR, Smart JE, Burridgel K, Helfmanll DM, and Thomas GP. "Co-existence of
Vinculin and a Vinculin-like Protein of Higher Molecular Weight in Smooth Muscle". J. Biol.
Chem. 257 (18): 11024-11031; 1982.
15."General remarks on handling RNA". The Bench Guide. Qiagen.
16.Grassman W and Deffner G. “Verteilungschromatographisches Verhalten von Proteinen
und Peptiden in phenolhaltigen Lösungsmitteln”. Hoppe-Seylor’s Z. Physiol. Chem. 293: 89-
98; 1953.
17.Kedzierzki W and Porter JC. "A Novel Non-Enzymatic Procedure for Removing DNA
Template from RNA Transcription Mixtures". Biotechniques 10 (2); 1991.
18.Kirby KS. "A New Method for the Isolation of Ribonucleic Acids from Mammalian Tissues".
Biochem. J. 64, 405; 1956.
19.Leonard DGB. Molecular Pathology in Clinical Practice. 1st edition. New York: Springer,
2007.
20.McMurry J. Organic Chemistry. 6th edition. Brooks Cole; 2003.
21.O'Neil M. The Merck Index. 14th edition. New Jersey: Merck & Co.; 2006.
7
22.Palmiter RD. "Magnesium precipitation of ribonucleoprotein complexes. Expedient
techniques for the isolation of undegraded polysomes and messenger ribonucleic acid".
Biochemistry, 13: 3606–3615; 1974.
23.Perry RP and Kelley DE. "The Production of Ribosomal RNA from High Molecular Weight
Precursors". J. Mol. Biol., 70: 265-279; 1972.
24.Perry RP, La Torre J, Kelley DE, Greenberg JR. "On the lability of poly(A) sequences during
extraction of messenger RNA from polyribosomes". Biochim Biophys Acta. 262 (2): 220-6;
1972
25.Puissant, C. and L.M. Houdebine. "An Improvement of the Single-Step Method of RNA
Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction". BioTechniques
8: 148 - 149; 1990.
26.Revest P and Longstaff A. Molecular Neuroscience. New York: Springer; 1998.
27.Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd
edition. New York: Cold Spring Harbor Laboratory Press; 1989.
28.Stenesh J. Dictionary of Biochemistry and Molecular Biology. 2nd edition. New York: John
Wiley & Sons; 1989.
29.Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter WJ and Goodman HM. "Rat insulin
genes: construction of plasmids containing the coding sequences". Science 196 (4296):
1313-1319; 1977.
30.Volkin E and Carter CE. “The Preparation and Properties of Mammalian Ribonucleic
Acids”. J. Am. Chem. Soc., 73 (4): 1516–1519; 1951.
31.Wallace D. "Large- and Small-Scale Phenol Extractions". Methods Enzymol., 152; 1987.