Download Optimization of the RT-PCR Method Using the TitanTM One Tube

T E C H N I C A L
T I P S
Kirsten Delp, Dirk Jung, and Barbara Seliger
III. Medizinische Klinik, Abteilung für Hämatologie/Onkologie, Langenbeckstraße 1,
D-55101 Mainz
Tel.: (+49) 61 31/17 6760, Fax.: (+49) 61 31/17 6678
Optimization of the RT-PCR Method Using the TitanTM One
Tube RT-PCR System
Introduction
RT-PCR is a highly sensitive method for
determining gene expression at the RNA
level and for quantifying the strength of
gene expression (i.e., the quantity of mRNA
[1], using only small amounts of material).
In addition to its use in numerous applications, RT-PCR can serve as a basis for identifying mutations and polymorphisms and
also facilitate the cloning of rare transcripts
while avoiding the costly preparation of
cDNA libraries.
In this article we compare the sensitivity
of various RT-PCR methods and analyze the
housekeeping gene β-actin that is strongly
expressed in all cells and the costimulatory
molecule B7-1 that is absent or weakly
expressed in tumor cells. β-actin and B7-1
were amplified both alone and in combination. Total cellular RNA from two human cell
lines, the T-lymphoma cell line T2 and the
pancreatic carcinoma cell line Panc1, were
employed for RT-PCR analyses. The results
show that the use of the TitanTM One Tube
RT-PCR System (Boehringer Mannheim)
increases the sensitivity of the RT-PCR reaction many times compared to the standard
RT-PCR method and also allows reproducible expression analysis of various genes at
the same time.
Material and Methods
The standard RT-PCR method:
“two-step” RT-PCR
To amplify RNA by the PCR method, a
primer is first hybridized to the RNA
matrix. A cDNA copy created with reverse
transcriptase can then be amplified through
a subsequent PCR reaction (3). Depending
on the experimental objective, various
sources of reverse transcriptase are available
for synthesizing the first cDNA strand (e.g.,
the enzyme of the monkey myeloblastosis
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NO. 4 [1997]
virus [AMV] or the Moloney mouse leukemia virus [M-MuLV]). The resulting cDNA
can then be duplicated using a heat-stable
DNA polymerase. In this method the reaction tube has to be opened after the cDNA
synthesis for the addition of the reagents
necessary for the cDNA amplification.
Thus, the conventional RT-PCR analysis is a
“two-step” method.
Reverse transcription procedure
1000 ng RNA
6.5 µl dNTPs (2 mM)
4 µl 5 x RT-PCR buffer
2 µl DTT (100 mM)
2 µl hexanucleotide mixture (Boehringer
Mannheim)
H2O to 18 µl
To denature the RNA heat the reaction
mixture to 95°C for 4 min and add the
following:
1 µl RNAse inhibitor
1 µl reverse transcriptase (10 U/µl)
Carry out the reverse transcription in a thermocycler using the following conditions:
22°C for 10 min
42°C for 40 min
95°C for 4 min
4°C endlessly
The cDNA obtained in this reaction is then
used for the subsequent RT-PCR.
RT-PCR procedure
Transfer 2 µl cDNA (= 100 ng total
RNA) from the reverse transcription to the
reaction tubes, add H2O to a final reaction
volume of 25 µl and prepare a mixture for
the PCR reaction that contains the following
component mix per sample:
0.5 µl 5'-primer (50 pmol)
0.5 µl 3'-primer (50 pmol)
4.0 µl MgCl2
5.0 µl Taq DNA polymerase buffer (10x)
5.0 µl dNTPs (2 mM)
1.5 µl Taq DNA polymerase buffer (1 U/µl)
H2O to 25 µl
Carry out the PCR reaction in the thermocycler using the following conditions:
95°C for 1 min (denaturation)
53°C for 1 min (annealing temperature for
β-actin/B7-1 primer
72°C for 1 min (DNA synthesis)
4°C endlessly (termination of the reaction)
Repeat these steps 30x.
Modification of the standard RT-PCR:
the “one-step RT-PCR”
In this method both the cDNA synthesis
and the amplification are performed with an
optimized buffer and the respective enzyme
one after the other, but without any more
addition of reagents. A distinction is made
between two approaches:
a. The use of T. thermophilus-(Tth-)DNA
polymerase which, like reverse transcriptase, is active in the presence of manganese and, moreover, accepts both RNA
and DNA as matrix, enables the whole
reaction to be performed as a “one-step”
RT-PCR analysis (4). The drawbacks of
this experimental approach include the
amplification of fragments only to a maximum of 1 kb and a relatively high error
rate for DNA polymerase as a result of the
manganese ion concentration.
In view of these disadvantages this
method was not used.
b.The Boehringer Mannheim RT-PCR system was used for this approach. In addition to the conventional components, this
product contains an enzyme mix consisting of AMV reverse transcriptase, Pwo and
Taq DNA polymerase and a special buffer
containing DMSO. In contrast with the
“one-step” RT-PCR method described
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T E C H N I C A L
T I P S
Primer
Length
Sequence
GC%
Annealing
temp.
Fragment
size
β-actin3'
21 bases
5'-GAAGCATTTGCGGTGGACCAT-3'
52
57°C
315 bp
53°C
1022 bp
β-actin3'
21 bases
5'-TCCTGTGGCATCCACCAAACT-3'
52
hB71cd3'
20 bases
5'-GGACGCTACCTTCAGATCTT-3'
50
hB71cd3'
20 bases
5'-AGCAATTGGATTGTCATCAG-3'
40
Table 1. Descriptions of primers.
above, this recently developed Titan One
Tube RT-PCR System offers more sensitive
detection of RNA molecules and amplification of RT-PCR products with lengths of
more than 2 Kb. The error rate during
PCR is reduced due to the Pwo DNA polymerase, and in addition, the use of the
DMSO-containing buffer reduces the secondary RNA structures that can adversely
affect the PCR analysis.
Master Mix 1/per sample:
5.0 µl dNTPs (2 mM)
1.5 µl 5' primer B7-1 (15 pmol)
1.5 µl 3' primer B7-1 (15 pmol)
1.5 µl 5' primer β-actin (1.5 pmol)
1.5 µl 3' primer β-actin (1.5 pmol)
100 ng template RNA
2.5 µl DTT (100 mM)
0.25 µl RNAse inhibitor
H2O to 25 µl
315 bp
β-actin
M
T2
Panc
β-actin
B7-1
T2
Panc
β-actin
M
T2
Panc
B7-1
Figure 1 Agarose gel electrophoresis of the PCR products from T2 and Panc1 RNA with the conventional
“two-stage” RT-PCR method using β-actin and B7-1 primers alone and in combination. M: 100 bp
molecular weight marker.
This method was used in order to
amplify both β-actin and B7-1 at the same
time in a single reaction. Furthermore, the
sensitivity of the RT-PCR method was determined by using different concentrations of
RNA for the reaction.
The studies employed total cellular RNA
from T2 cells and the test protocol developed by Boehringer Mannheim.
As described by Boehringer Mannheim,
two different reaction components are prepared: Master Mix 1 and Master Mix 2:
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Master Mix 2/per sample:
10 µl RT-PCR buffer (5x) containing
1.5 mM MgCl2
1.0 µl enzyme mix
H2O to 25 µl
Pipette 25 µl portions of Master Mix 1 and 2
into a thin-walled PCR reaction tubes. Incubate the solution for 30 min at 50°C.
Carry out the PCR reaction in the thermocycler using the following conditions:
94°C for 2 min (denaturation of the
template)
94°C for 30 s
53°C for 30 s (primer annealing)
68°C for 1 min (extension time)
Repeat these cycles 10x.
94°C for 30 s (denaturation of the template)
53°C for 30 s (primer annealing)
68°C for 1 min + 5 s per cycle (extension
time)
Repeat these cycles 25x.
68°C for 2.5 min
4°C endlessly (termination of the reaction).
Results and Discussion
Total cellular RNA from T2 and Panc1
tumor cells was isolated by the guanidine
isothiocyanate-cesium chloride centrifugation method (2) and reverse-transcribed
according to the test protocol as described in
“Materials and Methods,” “two-step” RT-PCR.
β-actin and B7-1, alone or in combination,
were then amplified using the specific primers
listed in Table 1. The PCR products were then
separated using a 2% agarose gel. As shown
in Figure 1, the use of β-actin-specific primers
resulted in the detection of a specific PCR
product of the correct size, whereas no signal
was obtained after amplification of the cDNA
with B7-1-specific primers. Furthermore, it
was not possible to amplify β-actin and B7-1
simultaneously using this method. These
results suggest that the conventional RT-PCR
method is not sufficiently sensitive for detecting the weak expression of B7-1 gene in T2
and Panc1 cells, whereas the “housekeeping”
β-actin gene that is strongly expressed ubiquitously could be amplified without difficulty.
As shown in Figure 2, the corresponding PCR product of the correct size could be
detected in T2 and Panc1 tumor cells in the
“one-step” RT-PCR using the B7-1-specific
primer. To determine the sensitivity of the
“one-step” RT-PCR method, B7-1 and
β-actin were amplified simultaneously,
using different concentrations (1000 ng,
100 ng, 10 ng) of RNA from T2 cells. As
shown in Figure 3, 10 ng total cellular
RNA was sufficient to amplify both β-actin
and B7-1 in parallel.
In general, these investigations confirmed that the Titan One Tube PCR System
is capable of amplifying weakly expressed
genes, that β-actin can reproducibly be used
as an internal standard, and that the sensitivity is significantly increased compared to
the conventional PCR protocol. Consequently, this PCR method:
a. Can be used for semiquantitative analysis
of gene expression and
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T E C H N I C A L
T I P S
References
1. Brenner, C. A., Tarn, A.W., Nelson, P. A., Engelman,
E. G., Suzuki, N.,Fry, K. E., and Larrick, J. W. (1989)
Biotechnique 7: 1096.
2. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J.,
and Rutter, W. J. (1979) Biochem. 18: 5294.
3. Kawasaki, E. S. in PCR Protocols, ed. Innis, M. A.,
Gelfand, D. H., Sninsky, J. J., and White, T. J., Academic Press. San Diego (1990), p. 21.
4. Myers, T. W., Geiland, D. H. (1991) Biochem. 30:
7661.
1022 bp
B7-1
M
T2
Pane
Figure 2 Agarose gel electrophoresis of the B7-1
PCR products obtained with the TitanTM One Tube PCR
System using 100 ng total RNA for the analysis.
M: 100 bp molecular weight marker.
b. Due to the low RNA concentrations
required - further used for screening
experiments where only little material/
RNA is available.
1022 bp
B7-1
315 bp
β-actin
M
1000 ng
100 ng
10 ng
Figure 3 Agarose gel electrophoresis of the B7 -1
PCR products obtained with the TitanTM One
Tube PCR System using differing concentrations of total RNA (1000 ng, 100 ng, 10 ng) for
the analysis. M: 100 bp molecular weight marker.
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NO. 4 [1997]
Product
TitanTM One Tube
RT PCR System*
Cat. No.
Pack Size
1 888 382 25 reactions
1 855 476 100 reactions
Hexanucleotide Mix
1 277 081
100 µl
(50 reactions)
Also Available
Taq DNA Polymerase,
5 units/µl*
Cat. No.
1 146 165
1 146 173
1 418 432
1 596 594
1 435 094
Pack Size
100 units
500 units
4x250 units
10x250 units
20x250 units
Pwo DNA Polymerase*
1 644 947
1 644 955
100 units
2x250 units
Reverse Transcriptase
AMV
1 495 062
109 118
500 units
1000 units
Reverse Transcriptase
M-MuLV
1 062 603
500 units
* Purchase of these products is accompanied by a limited
license to use them in the Polymerase Chain Reaction (PCR)
process in conjunction with a thermal cycler whose use in the
automated performance of the PCR process is covered by the
up-front license fee, either by payment to Perkin-Elmer or as
purchased (i.e., an authorized thermal cycler).
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