T O O L S
TRIZOL™: A NEW REAGENT FOR OPTIMAL SINGLE-STEP
ISOLATION OF RNA
P
rogress in many fields of basic and
clinical research depends on the avail-
ability of high-quality RNA. Of the
many methods for isolating RNA
(reviewed in 1 and 2), a single-step RNA isola-
tion method based on guanidine isothio-
cyanate/phenol/chloroform extraction (3) has
been growing in popularity because it dramati-
cally reduces the amount of time required to
isolate RNA without sacrificing its quality.
TRIZOL™ Reagent is an improved version of
the single-step RNA isolation method.
TRI ZOL Reagent (patent pending) is a
monophasic solution of phenol and guanidine
isothiocyanate. During sample homogenization
or lysis, TRIZOL Reagent maintains the integri-
ty of the RNA, while disrupting cells and solu-
bilizing cell components. Addition of
chloroform, followed by centrifugation, sepa-
rates the solution into an aqueous phase and an
organic phase. The RNA remains in the aque-
ous phase and is recovered by precipitation with
isopropanol. The DNA and proteins remain in
the organic phase and can be recovered by
sequential precipitation (4). Precipitation with
ethanol yields DNA from the interphase, and an
RNA Ladder
0.24–9.5 kb
1 2 3 4 5 complexes, 0.2 ml of chloroform was added per
FIGURE 1. Gel analysis of total RNA recovered from
tissues and cells using TRIZOL Reagent. Two micrograms
of RNA were loaded per lane on a 1.5% agarose-formalde-
hyde gel. Lane 1, rat pancreas. Lane 2, calf thymus. Lane 3,
rat brain. Lane 4, rat liver. Lane 5, HeLa cells.
F O C U S 1 5 N U M B E R 4 99
additional precipitation with isopropanol yields Domenica Simms
proteins from the organic phase (4). Molecular Biology
This technique can be used with small Research and
quantities of tissue (50 to 100 mg) and cells Development
(5 × 106) and large quantities of tissue (≥1 g) Life Technologies, Inc.
and cells (>107), of human, animal, plant, or Gaithersburg,
bacterial origin. The simplicity of the TRIZOL Maryland 20884
Reagent method allows simultaneous process-
ing of a large number of samples. The entire Paul E. Cizdziel
procedure can be completed in one hour. The Life Technologies
RNA can be used for Northern blot analysis, Training Center
dot blot hybridization, poly (A)+ selection, in Germantown,
vitro translation, RNase protection assay, Maryland 20876
molecular cloning, and polymerase chain
reaction (PCR). Piotr Chomczynski
Department of
METHODS Internal Medicine
Isolation of total RNA. Tissue samples were School of Medicine
homogenized in GIBCO BRL TRIZOL Reagent University of
(Cat. No. 15596; 1 ml per 50 to 100 mg tis- Cincinnati
sue) using a glass-TEFLON® or power homoge- Cincinnati, Ohio
nizer (Polytron, Tekmar TISSUMIZER®). 45267
Cells grown in suspension (HeLa and
Jurkat) were collected by centrifugation.
Human lymphocytes and leukemic cells
(5 × 10 6 to 1 × 10 7) were isolated on FICOLL-
H Y P A Q U E ® and lysed in 1 ml of TRI Z O L
Reagent by repetitive pipetting.
After incubation of the homogenized sam-
ples for 5 min at room temperature to permit
the complete dissociation of nucleoprotein
1 ml of TRIZOL Reagent. The samples were
mixed vigorously and then centrifuged at
12,000 × g for 15 min at 4°C. Centrifugation
separated the biphasic mixtures into the lower
red, phenol-chloroform phase and the upper
colorless, aqueous phase.
The RNA was precipitated from the aque-
ous phase by mixing with 0.5 ml of isopropanol
(for each initial milliliter of TRIZOL Reagent).
The samples were incubated at room tempera-
ture for 10 min and centrifuged at 12,000 × g
for 10 min at 4°C. The supernate was removed
and the RNA pellet was washed once with
75% ethanol. The pellet was air dried and
dissolved in diethyl pyrocarbonate (DEPC)-
 treated water. The RNA was quantitated by
A260 measurement.
Northern blot. Total RNA (2 µg or 5 µg
each) was electrophoresed on agarose-formalde-
hyde gels in MOPS buffer and transferred to
nylon membranes. The membranes were prehy-
bridized for 1 h and then hybridized overnight
at 42°C in 50% (v/v) formamide, 0.9 M NaCl,
60 mM NaH 2 PO 4 , 6 mM EDTA, 5X
Denhardt’s solution, 1% SDS, 200 µg/ml dena-
tured herring sperm DNA, 10% dextran sulfate,
and 200 ng of denatured biotinylated-DNA
probe per milliliter. The 2.3-kb, cloned BamH I
to Hind III fragment of the human HSP70
gene (5), used as DNA probe, was biotinylated
using the G IBCO BRL B IO N ICK ™ Labeling
System. Presence of human HSP70 mRNA was
detected by chemiluminescence using the GIBCO
BRL PHOTOGENE™ Detection System.
S1 nuclease analysis. The analysis was per-
formed using a portion of the human HSP70
gene as a probe. The Ava I to Hind III frag-
ment from the 3' region of the HSP70 gene
that contained the polyadenylation signal
(AATAAA) (6) was subcloned into pSPORT 1
DNA. The Ava I DNA fragment was labeled
using the large fragment of E. coli DNA poly-
merase I, in a reaction containing 0.75 µM
1 2 3 4
FIGURE 2. Northern blot. Detection of a ~2.2-kb tran-
script in RNA isolated with TRIZOL Reagent from heat-
shocked human HeLa cells, using a human HSP70
biotinylated probe and chemiluminescence (exposure time
20 min). Lane 1, 5 µg of heat-shocked HeLa cells RNA.
Lane 2, 5 µg of Jurkat cells RNA. Lane 3, 2 µg of heat-
shocked HeLa cells RNA. Lane 4, 2 µg of Jurkat cells RNA.
100
[α-32P]dCTP (400 Ci/mmol), 750 µM dGTP,
20 mM Tris-HCl (pH 8.0), 40 mM NaCl,
9 mM MgCl2, and 1 mM DTT. The radiola-
beled probe DNA was purified using the GIBCO
BRL G L A S S MAX™ DNA Isolation Spin
Cartridge System. RNA (5 µg) and probe DNA
(5 × 104 cpm) were hybridized overnight at
56°C and then digested with 300 units of S1
nuclease for 1 h at 30°C (1). The samples were
electrophoresed on a 6% polyacrylamide-urea
gel prepared using GIBCO BRL GEL-MIX® 6.
RT-PCR. The SUPERSCRIPT™ Preamplifica-
tion System was used for first strand cDNA syn-
thesis from 1 µg of RNA (7,8). To determine if
the amplification product was exclusively from
the RNA, a control without reverse transcrip-
tase (RT) was occasionally included.
The PCR contained 1 to 20 µl of the first
strand cDNA reaction mixture, 0.1 µM each of
the suitable primer set, and 2.5 units of
AMPLITAQ™ DNA Polymerase (Perkin Elmer).
After an initial denaturation step at 94°C for
5 min, cycling conditions were: 30 cycles of
1 2 3 4 5 bases
—2,914
—1,175
—404
—327
—231
—141
—87
—54
FIGURE 3. S1 nuclease assay. Total RNA (5 µg) isolated
from Jurkat and heat-shocked HeLa cells using TRIZOL
Reagent was hybridized to a 3' 32P-end-labeled DNA frag-
ment of the HSP70 genomic DNA and treated with S1
nuclease as described in Methods. Lane 1, HeLa RNA. Lane
2, Jurkat RNA. Lane 3, yeast tRNA. Lane 4, 32P-end-
labeled Taq I fragments of φX174 (RF) DNA. Lane 5,
probe (no S1 nuclease).
F O C U S 1 5 N U M B E R 4
94°C for 45 to 60 s, 30 to 120 s at the appro-

100 bp
priate annealing temperature, and 72°C for 1 to

Ladder
DNA
3 min on a Perkin-Elmer DNA Thermal Cycler. 1 2 3
PCR reaction mixtures (10 µl each) were ana-
lyzed by electrophoresis on agarose gels and
stained with ethidium bromide.
In some cases, genomic DNA was removed
from RNA samples by treating total RNA with
GIBCO BRL DNase I, Amplification Grade.

RESULTS AND DISCUSSION

TRIZOL Reagent isolates large- and small-
size RNA species. The isolated RNA has an
A260/280 ratio of 1.6 to 1.8. Typical yields of
RNA per milligram of tissue are: liver and
spleen, 6 to 10 µg; kidney, 3 to 4 µg; skeletal
muscles and brain, 1 to 1.5 µg; placenta, 1 to
4 µg. The expected yield of RNA from 1 × 106
cultured cells is: epithelial cells, 8 to 15 µg;
fibroblasts, 5 to 7 µg.
Some tissues, like rat pancreas, are difficult
tissues from which to isolate intact RNA
because they are rich in nucleases. With other
tissues (e.g., calf thymus), RNA isolation is
plagued by persistent carryover of interphase
during transfer of the aqueous phase. TRIZOL
Reagent locks proteins, including nucleases,
into the interphase and organic phase, allowing
clean transfer of the RNA contained in the
aqueous phase. The recovery of RNA from calf
thymus was six times higher from TRIZOL than
from the original single-step method.
To assess the quality of the isolated RNA,
samples were resolved by gel electrophoresis
(figure 1). A typical pattern was obtained for
the RNA samples tested, indicative of unde-
graded RNA: sharp ribosomal RNA bands with
FIGURE 4. RT-PCR products from normal and leukemic cell total RNA isolated by
TRIZOL Reagent. Lane 1, normal lymphocytes, using primers for intact AML1 transcript.
Lane 2, normal lymphocytes using primers for the AML/CDR transcript. Lane 3, leukemic
cells using primers for the AML/CDR transcript. The primers used were: GAU AUA UCC
AUG GTC GAA GTG GAA GAG GGA and GAU UAC UUA GAG GCT GAG GGT
TAA AGG in lane 1; and (CUA)4TGT CGG TCG AAG TGG AAG AGG and (CUA)4CTT
GAG TAG TTG GGG GAG GTG G in lanes 2 and 3.
previous reports (350-bp protection product) is
not known. In addition, a protection product of
75 bases was observed. The nature of this 75-
base protection fragment is unknown.
However, it was highly reproducible and may
be generated by an alternative polyadenylation
event, protection from a related but distinctly
different heat-shocked gene, or incomplete
denaturation during electrophoresis. No protec-
tion fragments were observed in S1 nuclease
analysis with RNA isolated from Jurkat cells.
RNA samples isolated using TRI Z O L
Reagent can be used directly as substrates for
RT-PCR when primers span more than one
100 bp
Ladder

the 28S band about two-fold more intense than

DNA

the 18S band. 1 2 3 4 5

Northern blot analysis was performed to
verify the integrity of unique mRNAs and their
ability to hybridize in membrane-based assays.
The presence of the 2.2-kb human heat shock
protein HSP70 mRNA was detected in the
heat-shocked HeLa cells, but not in the control
Jurkat cells (figure 2).
S1 analysis of RNA from heat-shocked
HeLa cells revealed that polyadenylation
occurred at ~330 bp downstream from the Ava FIGURE 5. β-actin RT-PCR product from rat pancreas total RNA isolated by
I site (figure 3). This is close to the site of TRIZOL Reagent. Lane 1, primers reaction mixture. Lane 2, RNA, RT present. Lane 3,
polyadenylation previously reported (5). The RNA, no RT present. Lane 4, RNA treated with DNase I, RT present. Lane 5, RNA treated
with DNase I, no RT present.
reason for the 20-bp discrepancy in size from

F O C U S 1 5 N U M B E R 4 101
 exon. RNA from human normal lymphocytes
and leukemic cells that contain a characteristic
translocation, the t(8;21), were used for RT-
PCR (9). The translocation results in the gener-
ation of a fusion gene, AML/CDR. A 251-bp
fragment representing the junction region
between AML1 and CDR was present in t(8;21)
positive leukemic cDNA but not in normal lym-
phocytes, which instead contain an intact AML1
gene detected as a 320-bp fragment by using
specific primers (figure 4).
For rat pancreas RNA isolated using TRIZOL
Reagent, treatment with DNase I, Amplification
Grade, was required prior to RT-PCR to obtain
the β-actin amplification product (figure 5).
SUMMARY
TRIZOL Reagent was developed for obtain-
ing high-quality total RNA in high yield. Total
RNA isolated with TRIZOL Reagent is suitable
for common RNA applications such as Northern
blots, S1 nuclease assays, RT-PCR, and poly(A)+
selection. The method is fast, reliable, and sim-
plifies simultaneous processing of many samples.
ACKNOWLEDGEMENT
We would like to thank Dr. Nicoletta Sacchi
[Department of Biology and Genetics, School of
Medicine, University of Milan, Italy] for pro-
viding the RT-PCR data from human normal
lymphocytes and leukemic cells—and for her
critical reading of the manuscript.
REFERENCES
1. Sambrook, J., Fritsch, E.F., and Maniatis, T.
(1989) Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
2. Ausubel, F.M., Brent, R., Kingston, R.E., Moore,
D.D., Seidman, J.G., Smith, J.A., and Struhl, K.,
eds. (1989) Current Protocols in Molec. Biol., Vol.
1, Greene Publishing Assoc. and Wiley
Interscience, New York, 4.0.1.
3. Chomczynski, P. and Sacchi, N. (1987) Anal.
Biochem. 162, 156.
4. Chomczynski, P. (1993) Biotechniques, 15, 532.
5. Wu, B., Hunt, C., and Morimoto, R. (1985) Mol.
Cell. Biol. 5, 330.
6. Hunt, C. and Morimoto, R. (1985) Proc. Natl.
Acad. Sci. USA 82, 6455.
7. Hu, A.W., D’Alessio, J.M., and Kullman, J.
(1991) FOCUS 13, 130.
8. Nisson, P.E., Rashtchian, A., and Watkins, P.C.
(1991) PCR Methods Appl. 1, 120.
9. Nisson, P.E., Watkins, P.C., and Sacchi, N.
(1992) Cancer Genet. Cytogenet. 63, 81.

Ask The GIBCO BRL How large a tissue sample can be used with
TRIZOL™ Reagent?
The TRIZOL Reagent can purify RNA from up to
T ECH-L INESM 100 mg of tissue. The sample volume should not
exceed 10% of the volume of TRIZOL Reagent
used. If poor phase separation is observed, add
an additional half-volume of TRIZOL Reagent.

102 F O C U S 1 5 N U M B E R 4