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Epicentre Forum 5 (4)
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Judith T. Schanke, Epicentre Technologies
Introduction Multiplex RT-PCR (also referred to as relative RT-PCR) is
commonly used for the semi-quantitative analysis of gene expression levels
when defining tissue-restricted gene expression patterns. Typically,
multiplex RT-PCR is performed to determine the changes in expression
level of a gene in a series of tissue types, throughout stages of development
or cellular differentiation, or after specific experimental treatments.
Multiplex RT-PCR is also commonly used to examine the expression patterns
of a series of related genes and to look at various regions of a large
message for mutation analysis.3-6 Using this method, comparisons
of specific RNA target levels among a series of samples can be made in
relation to the amount of product obtained from a co-amplified, internal
standard. Semi-quantitative results are typically expressed as a percent
or fold difference in the level of a specific RNA between otherwise equivalent
RNA samples. The amplification of an internal standard in these reactions
provides a means of controlling for RNA integrity and the quantity of
RNA added to the assay.
Here, we use multiplex RT-PCR with the MasterAmp™ RT-PCR Kit to
examine the tissue-restricted expression of the human chorionic gonadotropin (CG) alpha
subunit. The appearance of CG provides early confirmation of pregnancy
in higher primates. The physiological role of CG is to stimulate the
corpus luteum to secrete progesterone, maintaining the implanted embryo
until the onset of placental steroid hormone secretion.1 CG
is a heterodimer composed of alpha and beta subunits. The single-copy
alpha subunit gene encodes a common component of all glycoprotein hormones.
The alpha gene is expressed in pituitary gonadotropes and thyrotropes
of all mammals and in placental trophoblasts of primates and horses.2 Expression
of CG beta is restricted to the placental trophoblast.
Using the MasterAmp RT-PCR Kit, the linear range of CGa amplification
was determined, CGa and the housekeeping gene GAPDH were co-amplified,
and the expression pattern of CGa in a series of human cellular RNA samples
were compared. The MasterAmp RT-PCR Kit was well suited for multiplex
amplifications. The high-temperature reverse transcription, inclusion
of MasterAmp PCR Enhancer (with betaine)*, and the convenient
one-tube format assured efficient, reproducible multiplex amplifications.
There are a number of critical parameters in designing multiplex RT-PCR
experiments, including primer design. Because of the increased number
of primers present in the reaction, it is important to design primers
that minimize dimer and secondary structure formation. It is also important
to design the primers with relatively high, comparable melting temperatures
to minimize non-specific amplifications. High-temperature reverse transcription
with a thermostable RT DNA Polymerase like RetroAmp™ RT Polymerase
and specific primers also minimizes non-specific amplification.
Comparison of message levels in multiplex RT-PCR is made in the linear
range of amplification, where amplification efficiency is highest and
remains constant. At that stage, the product is still accumulating at
its maximum rate. Therefore, pilot experiments should be performed to
determine the amount of RNA template to use and the number of amplification
cycles to perform to assure analysis in the linear range.
Another critical parameter is the choice of an RNA standard. Ideally,
the standard should not be affected by the experimental treatment being
used, and the expression pattern of the standard should not change among
the tissue types and stages of development being examined. Some of the
most common amplification standards used in multiplex RT-PCR are the
following housekeeping genes; ß-actin, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and cyclophilin. Each of these genes is
expressed in a broad range of cell types and expression levels are maintained
across cell types and throughout various stages of cellular differentiation.
Unfortunately, most of these commonly used standards do vary in expression
with some experimental treatments and among some tissue types.7-11 Therefore,
careful review of the literature and pilot experiments verifying the
constant expression of the standard are needed.
RT-PCR reactions
The primer sequences used to amplify regions of both human CGa and GAPDH
messages were:
CGaF, 5'-GAGAACTCACAAGACGAAGCTAAAATCCC-3'
CGaR, 5'-CTCACAAAGCCATAAACACTGAACAAG-3'
hGAPDHF, 5'-TTCATTGACCTCAACTACAT-3'
hGAPDHR, 5'-GTGGCAGTGATGGCATGGAC-3'
MasterAmp RT-PCR reactions contained 100 ng of total cellular RNA, 12.5
pmoles of each primer, 1X RT-PCR buffer, 3 mM MgCl2, 1X MasterAmp
PCR Enhancer (with betaine), 0.5 mM MnSO4, 400 mM
each dNTP, and 2.5 units of RetroAmp RT DNA Polymerase. Samples were
typically amplified with the following cycling profile: reverse transcribe
at 60°C for 20 minutes, then amplify for 25 cycles of 92°C for 30 seconds,
56°C for 40 seconds, and 72°C for 60 seconds, with a final extension
of 4 minutes at 72°C.
Easy identification of the linear range of amplification
The linear range of amplification for a 589 bp region of hCGa was determined
by amplifying the region from total human placental RNA. A master mix
reaction was assembled with the MasterAmp RT-PCR Kit, and identical 50 µl
aliquots were pipetted into a series of tubes. Individual reactions were
removed from the thermal cycler after increasing numbers of cycles (22
to 34 cycles), and were extended for a final 4 minutes at 72°C. Five
microliters of each sample were separated by agarose gel electrophoresis,
stained with ethidium bromide, and visualized by transillumination (Figure
1). The range where amplification efficiency was at its peak and
product accumulated at its highest, constant rate was determined. The
linear range for this amplification was defined as 22-28 cycles.
 |
Figure 1. Linear range of CGa amplification. CGa amplifications
from total human placental RNA for 22 to 34 cycles. M = 100 bp
ladder. |
No optimization required for multiplex RT-PCR with the MasterAmp
Kit
Next, we performed simultaneous amplification of hCGa and hGAPDH from
human placental RNA. First, individual amplifications of hGAPDH and hCGa
were performed. Figure 2 shows that RT-PCR amplification of hGAPDH using
standard reaction conditions with 25 cycles produced a prominent band
at 443 bp. The hCGa amplification resulted in the 589 bp product using
the conditions optimized above. Multiplex RT-PCR was performed using
standard reaction conditions, but included the primers for both hGAPDH
and hCGa amplifications. Twenty-five cycles of PCR were performed using
the amplification profile described above. The multiplex amplification
of hCGa and hGAPDH shows both the 589 bp and 443 bp products (Figure
2).
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Figure 2. Multiplex amplification of hCGa and hGAPDH from placental
cellular RNA. Lane 1, hGAPDH amplification; Lane 2, hCGa
amplification; and Lane 3, co-amplification of hGAPDH and hCGa.
M = 100 bp ladder. |
The expression of hCGa is cell-type restricted
To analyze the hCGa expression levels in various tissues, multiplex
amplifications of hCGa and hGAPDH were performed using the conditions
above with the MasterAmp RT-PCR Kit and human placental, lung, and heart
total cellular RNA. The results are shown in Figure 3. The hGAPDH amplification
product is present in all samples analyzed and served as a positive control
for amplification and RNA integrity. hCGa is clearly expressed at high
levels in placental RNA, but not in lung or heart samples. The additional
amplification products detected when heart RNA served as a template were
present in amplifications containing hGAPDH primers alone, suggesting
they are not hCGa related (data not shown). GAPDH pseudogenes
are present throughout the human genome.12 These additional
products probably resulted from the amplification of expressed pseudogenes,
as previously described in human liver.13
| Figure 3. Multiplex RT-PCR of hCGa and hGAPDH messages in total
cellular RNA. Co-amplification of RNA isolated from placenta
(Lane 1), lung (Lane 2), and heart (Lane 3). M = 100 bp ladder.
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The MasterAmp RT-PCR Kit, with RetroAmp RT DNA Polymerase and MasterAmp
PCR Enhancer (with betaine), was convenient and effective in
multiplex RT-PCR to examine the tissue restricted expression pattern
of CGa. Multiplex RT-PCR requires special attention to experimental design
including careful primer design, preliminary experiments to define the
linear range of amplification, and prudent choice of RNA standard. High-temperature
reverse transcription, with well designed specific primers and a thermostable
enzyme like RetroAmp RT DNA polymerase, minimizes secondary structure
interference and non-specific amplification products during multiplex
amplifications. The convenience of one-step, one-tube amplifications
with the MasterAmp RT-PCR Kit allowed quick definition of the linear
range, as well as rapid examination of entire sets of multiplex reactions.
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MasterAmp™ High Fidelity RT-PCR Kit
MasterAmp™ RT-PCR Kit for High Sensitivity
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