 Competitive RT-PCR vs. real-time RT-PCR Literature:
RT-PCR & real-time RT-PCR (all)
Literature: classical block
RT-PCR & competitive RT-PCR
Introduction
The theory is straightforward,
but a number of technical caveats are associated with the use of conventional end-point
methodologies for quantitative RT-PCR. In these techniques, PCR results are monitored after
a given number of cycles, by which point factors such as limiting reagent
concentrations and side reactions may have played a significant
role in effecting final product concentration. Quantitative
competitive PCR was developed in response to some of these
difficulties. In this approach, the starting amount of target is calculated based on the ratio
of
target to competitor after amplification. However, quantitative
Real time PCR or real-time
RT-PCR offers numerous advantages over previous attempts at quantitating
RT-PCR. Other methods typically rely on end-point measurements, when often the
reaction has gone beyond the exponential phase because of limiting
reagents. To compensate for such problems, competitive PCR was devised, which allows for
normalization of the end product based on the ratio between target and
competitor. Because this method is cumbersome, requiring a carefully
constructed competitor target for each (RT-)PCR reaction and a series of dilutions to ensure
that there is a suitable ratio of target to competitor, it is seldom used successfully.
In contrast, with real time (RT-)PCR, the dynamic range is much greater than that
of competitive (RT-)PCR - up to 8 orders of magnitude as compared to one
with competitive (RT-)PCR -, post-reaction processing is eliminated, and
the measurements are taken from the exponential range of the reaction, where
component concentrations are not limiting. And best of all, the entire
process is automated.
Slide show competitive RT-PCR vs. real-time RT-PCR Poster competitive RT-PCR Poster real-time PCR Poster Boards
Competitive
RT-PCR
by D Sugden (Endocrinology & Reproduction Research Group, King's College, London) Molecular Endocrinology
Workshop 10th November 1999
Competive
PCR Guide
by TaKaRa Biochemicals Principales and Difficulties of competitive PCR 
a omparison of primer-dropping, competitive, and real-time RT-PCRs Wall SJ, Edwards DR. Anal Biochem 2002 Jan 15;300(2):269-73
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. Although all three
types of quantitative RT-PCR produce a similar mRNA expression
profile, the real-time and competitive RT-PCR methods are the most reliable
as they produce
significantly similar results. Of these techniques real-time RT-PCR is
most favorable mainly due to the easier methodology. Niels
Wedemeyer, Thomas Pötter, Steffi Wetzlich, and Wolfgang
Göhde Background: Competitive PCR
of reverse transcribed mRNA sequences is used to quantify transcripts,
but the usual approaches are labor-intensive and time-consuming. We
describe the non-gel-based quantification of competitive reverse
transcription (RT)-PCR products with use of microparticles and flow
cytometry.
Methods: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively. Results: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half. Conclusions: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.
Development and validation of an internally standardised competitive Reverse Transcription-Polymerase Chain Reaction (compRT-PCR) M. Pfaffl, H.H.D.
Meyer and H. Sauerwein (1998) M. Pfaffl; F.
Schwarz & H. Sauerwein (1998) Summary
<>To investigate the role of
local IGF-1 mRNA expression in various tissues, we developed and validated a method which
allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally
standardised Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A
synthetic competitive template IGF-1 standard cRNA (IGF-1 cRNA) was designed,
which contains the same flanking primer sequences used to amplify the wild type
IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration
present in tissue RNA samples, series of 250 ng total-RNA were spiked with
three
known quantities of the standard IGF-1 cRNA, incubated for competitive
RT-PCR
reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240
bp from the wild type IGF-1 mRNA) were subsequently separated and
quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R =
competitor PCR product / wild type PCR product) was plotted against the number of
starting molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA
present in the sample can then be read off where R = 1. The validated assay had a
detection limit of 1600 IGF-1 cRNA molecules/reaction, the
intra-assay
variation was 7.4% (n = 5) and linearity (r = 0.997) was given between
140 ng to
840 ng total-RNA input. The present method was first applied to study
the effect of long term castration on the IGF-1 expression rates in bovine
tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the
plasma concentrationsas quantified by RIA and were higher in intact
than in castrated animals. In two skeletal muscles (m. splenius
and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower
than in liver, respectively, without any differences between steers and bulls. In
bulls, the IGF-1 mRNA expression was higher in m. splenius (p<0.01) than m.
gastrocnemius, indicating that locally produced IGF-1 might be important for sexually
dimorphic muscle growth patterns.
IGF-1 mediates the anabolic
growth hormone actions in skeletal tissues. Above that locally expressed IGF-1 is an
important growth regulator acting in auto- and paracrine way (Thissen et al., 1994). To
investigate the tissue specific expression in low abundant tissues a method is required
which allows for a reliable quantification of IGF-1 mRNA. Considering these limitations,
RT-PCR offers the most potent instrument to detect low-abundance mRNAs and the
detection limit can been increased up to 1000-fold in comparison to other methods,
e.g. Northern hybridisation (Saiki et al., 1988). The relationship between the
initial
amount A of target mRNA present in the tissues and the amount Yn of DNA produced
after n PCR cycles can be expressed as
Yn = A *(1+E)n
where E is the amplification
efficiency of one reaction step (Chelly et al., 1988). Small variations in the
reaction
efficiency, therefore, translate into large differences in the amount of RT-PCR
product generated after n cycles. These limitations in quantitative analyses can be
compensated by parallel co-amplification of the native mRNA together with known amounts of
an internal standard cRNA. The amplification efficiency should affect both templates
similarly. Several designs have been used in quantitative RT-PCR to obtain an internal
standard cRNA that suits the characteristics of having an identical amplification
efficiency as the wild-type mRNA template and of being easy distinguishable from it
(Nedelman et al., 1992). Hereby the construction of an internal standard by inserting (Martini
et al., 1995) or deleting (Becker-Andrè and Hahlbrock,
1989; Piatak
et al., 1993; Malucelli et al., 1996) a relatively small sequence
within the wild type template are common practice. Due to the negative
relationship between the efficiency of amplification and the length of
the amplified sequences, the both templates should be as short as
possible (Rolfs et al., 1992). Analysis and quantification of
competitive
PCR products
can
be done either by electrophoretic separation with densitometric
quantification or by HPLC and following UV detection at 260 nm. HPLC-UV
is the most exact quantification method for PCR products in
terms
of accuracy, precision and linearity (Katz et al., 1990). In consequence we designed,
developed and validated an internally standardised IGF-1 mRNA RT-PCR assay with
subsequent HPLC-UV quantification for quantitative comparisons in tissues of low IGF-1 mRNA
abundance. The method was first applied to investigate the effect of castration on IGF-1
mRNA expression in bovine liver and two different skeletal muscles.
Establishment and Validation of the quantitative IGF-1 mRNA RT-PCR Assay conditions
Considering the
described criteria we designed a short internal standard IGF-1 cRNA,
for which
the
same flanking primers are used as for the wild-type IGF-1 mRNA. The
conditions
for the RT-PCR as described in Materials and Methods were optimised
with regard to PCR buffer pH, primer and MgCl2 concentration in the
PCR reaction, dNTPs concentration and annealing temperatures. To
ensure a parallel start in all individual reaction tubes and to increase specificity, yield
and precision of the PCR, a "hot-start" amplification with a melting wax barrier between RT
reagents and PCR master-mix was applied. The quantification of wild-type IGF-1 mRNA in
different tissues required a preliminary estimation of the IGF-1 cRNA start-molecule
concentration range to be used for individual tissues. This was performed by 7
titration steps from 1.6 * 108 to 1.6 * 1011 cRNA
start-molecules versus 250 ng total tissue RNA. For routine comparisons,
three standard concentrations covering the range in which
equal amounts of the two amplification products are to be expected for a certain tissue
were selected.
Amplification
efficiencies The amplification efficiencies
for the wild-type and the standard template were recorded during the exponential and the
plateau phase of the PCR. Figure 3a shows the results of the competitive
co-amplification for the two amplificates. Until cycle numbers 23-25
there was
an exponential increase in the amount of both products, followed by the
plateau phase. In order to compare the amplification efficiencies of target
IGF-1 mRNA and standard IGF-1 cRNA, the 10log of the HPLC
integrals (10log Yn) was plotted versus the number of PCR cycles (abscissa) and
the linear regressions were then calculated for the exponential and the plateau
phase (Figure 3b). The relationship Yn = A * (1+E)n,
in which
E is the amplification efficiency of interest, can be transformed to 10log Yn = n * 10log
(1+E) + 10log A, yielding a linear equation: y = x * a +
t. The
resulting efficiencies of competitive IGF-1 RT-PCR during the
exponential phase were nearly identical (Figure 3b; E (IGF-1 cRNA) =
0.66 ; r = 0.98 and E (IGF-1 mRNA) = 0.65 ; r = 0.98). Similarly, during the
plateau phase the amplification efficiencies were parallel with
E (IGF-1 cRNA) = 0.05 (r = 0.78) and E (IGF-1 mRNA) = 0.04 (r =
0.75). The initial ratio R of the both products remained constant throughout the
amplification cycles.
The sensitivity
of the RT-PCR was evaluated using different starting amounts of IGF-1
cRNA from
2.8 ag (16 IGF-1 cRNA molecules) to 28 ng (1.6 * 1011 cRNA molecules). The minimal detectable amount
of
IGF-1 cRNA using the HPLC-UV detection modus was 1600 molecules/tube.
Linearity and variability
The precision of the HPLC-UV
quantification of PCR products was initially established by quantifying 28 individual DNA
samples at 7 different concentrations from 5 to 325 ng DNA. A linear relationship
between the DNA concentration injected (d) onto the DEAE column and the respective peak
integral (i) could be demonstrated (i = 1.13 * d + 3.62; r
= 0.99). The
linearity of the RT-PCR was determined by quantifying the IGF-1 mRNA in
serial dilutions
of a liver RNA preparation (140, 280, 560 and 840 ng). Each RNA
dilution was assayed
together with four different IGF-1 cRNA standard concentrations. Figure
4 shows the
resulting ratio plots for the four individual RNA input concentrations.
The IGF-1 mRNA molecule numbers initially present were read off at R = 1.
In Figure 5 the amount of IGF-1 mRNA molecules (a) measured in
the different RNA dilutions is plotted versus the total-RNA input (t) into the RT-PCR
assay. A linear relationship between the amount of analyte and the measured IGF-1 mRNA
concentration could thus be demonstrated (a = 2.0 * 107 * t
+ 5.4 * 106; r = 0.997). To confirm the reproducibility
of the competitive IGF-1 RT-PCR, the assay
variation was determined: five identical RT-PCR experiments were set
up;
each with three different standard dilutions and 250 ng liver RNA.
Quantification resulted in 1.069 ± 0.079 * 109 IGF-1 mRNA molecules (n = 5) and thus in
an
assay variation of 7.4%.
Quantification of DNA,
total-RNA and IGF-1 mRNA in bovine tissues Table 1 summarises the DNA
concentration, total-RNA concentration and IGF-1 mRNA expression rates in liver and
in
two different muscles from steers and bulls. No significant changes in total
transcriptional activity
(total-RNA / DNA) within different tissue types could be observed. Hepatic IGF-1 mRNA
concentrations were higher in intact than in castrated males (p < 0.05) and were
correlated with the mean IGF-1 plasma concentrations recorded
one week before
slaughter (Figure 6). In bulls higher (p<0.01) IGF-1 mRNA
expression was observed in m. splenius than in m. gastrocnenius. In both
muscles the difference between bulls and steers did not reach the
level of significance (p = 0.07
for m. splenius and p = 0.22 for m. gastrocnemius, respectively).
RT-PCR is a potent and
sensitive methodology to detect low amounts of mRNA molecules and offers important insights
into the local expression system in low abundant tissues. Using competitive systems with
an internal standard, the limitations of quantitative power can be circumvented
(Becker-Andrè and Hahlbrock, 1989; Piatak et al., 1993; Martini et al., 1995). The
reliability of this approach depends on the condition of identical amplification efficiencies for
both, the wild-type and the standard RNA. Wang et al. (1989) postulated that
a reliable quantification of PCR products should remain within the
exponential phase
for both. In the IGF-1 mRNA quantification system described herein a
parallel co-amplification
of the two fragments could be substantiated, similarly as
reported by Bouaboula
et al. (1992) and Zimmermann and Mannhalter (1996) for other mRNA
species. As
demonstrated herein, the ratio between the two products remained
constant throughout the amplification, i.e. during the exponential and the
plateau phase. Thus the present IGF-1 mRNA quantification is not
necessarily limited to the exponential phase of the reaction. In view of the data provided
for
sensitivity, linearity and reproducibility, the competitive RT-PCR assay developed herein allows
for the absolute and accurate quantification of IGF-1 mRNA molecules with a sufficiently
high sensitivity even for tissues or cells with low abundancies or for very small amounts of RNA
available. We have first applied this IGF-1 mRNA quantification system to compare the IGF-1
expression rates in bovine tissues, however, the method can not only be applied in ruminant
tissues but also for comparisons in other species with sufficientlyhigh homologies of the
amplified IGF-1
fragment. Besides bovine tissues, we have successfully applied this method in porcine
tissues (Pfaffl et al., 1998). Considering the close relationship between the hepatic IGF-1 mRNA
concentrations and the IGF-1 plasma concentrations, a biological parallelism of IGF-1
mRNA transcription rates and IGF-1 protein translation might be postulated. The two
muscles investigated were selected according to their overproportional (m. splenius)
or underproportional (m. gastrocnemius) growth response to testicular steroids
(Brännäng, 1971). Castration divergently influenced the IGF-1
mRNA expression
rates in liver and in skeletal muscles. Comparing the IGF-1 mRNA
quantitiesin
the two investigated skeletal
muscle, we observed higher concentrations in m. splenius than in m. gastrocnemius in
bulls. Total transcriptional activity (RNA/DNA ratio) remained constant within all tissues and
the differences in IGF-1 mRNA were specific up- or down-regulations of the IGF-1
gene expression. These findings are in accordance with earlier investigations on
the higher growth impetus of m. splenius compared to m. gastrocnemius in bulls (Berg
and Butterfield, 1976). The molecular basis for this sexually dimorphic muscle
growth
pattern might be attributed to relativly higher sensitivities to testicular
steroids in neck muscle (Sauerwein and Meyer, 1989). Above that, the present study implies that
local differences in IGF-1 expression might be one of the mediators of the allometric
growth of these individual muscles in intact males.
insulin and thyroid hormone concentrations between two pig genotypes with markedly divergent growth rates and the effects of growth hormone treatment. Elsaesser, F.,
Pfaffl, M.W., Meyer, H.H.D., Serpek, B. and Sauerwein, H. (2002)
The intention of the current
study was to gain more insight into the endocrine and molecular controlmechanisms of growth in the
pig. For this purpose various growth related parameters
were determinedin 4-month-old barrows of two extreme pig genotyes, the
small,
obese Göttingen Miniature (GM) and thelarge and lean German Landrace
(DL). Mean growth hormone (GH) concentration, GH pulse frequencyand GH pulse amplitude did not
differ between breeds. Likewise, plasma IGF-1, thyroxine,tri-iodothyronine (T3)
concentrations were similar in both breeds. However the plasma GH
response(maximum
level and area under curve) to a single i.v. injection of GHRH in DL
was higher than in GM(P < 0.05). Furthermore, basal plasma insulin and in
particular plasma cortisol concentrations were higher in GM compared with DL
pigs (P < 0.05 and <0.01 respectively). Analysis of cortisol
during 4-hfrequent
blood sampling indicated higher cortisol amplitudes in GM compared with
DL (P < 0.01).Specific bGH-binding to hepatic membrane preparations was
not different between breeds and IGF-1 mRNA concentrations determined
by
reverse transcription-polymerase chain reaction in liver, m.semimenbranosus and m.
longissimus dorsi were similar in both breeds. I.m. treatment with
recombinant
porcine somatotropin (rpST; 70 mg/ kg live weight) over an 8-day period
in contemporary barrowsincreased without any breed difference, plasma IGF-1, T3 and
insulin concentrations and hepaticspecific bGH-binding, but did
not affect thyroxine or cortisol concentrations in plasma. IGF-1 geneexpression was also elevated in
liver and muscle tissues in rpST-treated animals without obvious breedeffects. The observations
underline the complexity of the hormonal and molecular control of
growth andsupport
the notion that differences in growth potential are the consequence of
differences at variouslevels of the somatotropic axis and apparently relate to
differences in other control systems of energymetabolism such as the
pituitary adrenal
axis or the endocrine pancreas as well.
by competitive co-amplification of a template in reverse transcription-polymerase chain reaction. Malucelli A,
Sauerwein H, Pfaffl MW, Meyer HHD.
We describe a polymerase chain
reaction (PCR)-based method for the quantification of androgen
receptor (AR) mRNA
in tissues. The amount of PCR products depends on the exponential
amplification of the
initial cDNA copy number; therefore minor differences in the efficiency
of amplification
may dramatically influence the final product yield. To overcome these
tube-to-tube differences
in reaction efficiency, an internal control AR cRNA was reverse
transcribed along
with the
target mRNA using the same primers. This standard was obtained by
deleting
a 38 bp fragment from an amplified bovine AR sequence, which was then
subcloned
and transcribed into cRNA. Known dilutions of the competitor
cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the
target mRNA. Following RT-PCR, the co-amplified specimens
obtained were
separated by gel electrophoresis and quantified by densitometric
analysis of ethidium bromide stain. We applied this method to quantify the
AR-mRNA in skeletal muscle of castrated as well as from intact
male cattle. The applicability of the quantification system for AR-mRNA
described herein was
demonstrated for other species, e.g. man.
Effects
of muscle type, castration, age, and compensatory growth
rate on androgen receptor mRNA expressionin bovine skeletal muscle.
Brandstetter AM,
Pfaffl MW, Hocquette JF, Gerrard DE, Picard B, Geay Y, Sauerwein H.
The effect of testosterone on
sexual dimorphism is evident by differential growth of forelimb and neck muscles in bulls and
steers. Divergent hormone sensitivites may account for the differential
growth rates
of individual muscles. Therefore, the objective of this study was to
compare
androgen receptor (AR) expression in three different muscles of bulls and
steers
at various ages and growth rates. Thirty Montbeliard bulls and 30 steers
were
assigned to four slaughter age groups. Four or five animals of
each sex were
slaughtered
at 4 and 8 mo of age. Animals in the remaining two slaughter groups (12
and 16 mo) were
divided
into groups of either restricted (R) or ad libitum (AL) access to feed.
Five animals
of each sex and diet were slaughtered at the end of the restricted
intake period at 12 mo of age. To simulate compensatory
growth, the remaining animals (R and AL) were allowed ad libitum
access to feed until
slaughter at 16 mo of age. Total RNA was
extracted from samples of semitendinosus (ST), triceps brachii (TB), and splenius (SP)
muscles. Androgen receptor mRNA was quantified in 200-ng total RNA
preparations using an internally standardized reverse transcription
(RT) PCR assay. Data
were analyzed using 18S ribosomal RNA concentrations as a covariable.
Steers had higher AR
mRNA
levels per RNA unit than bulls (P < .01). Androgen receptor mRNA
levels differed
between
muscles (P < .05), with lowest expression in the SP. The pattern of
AR expression
differed
(P < .05) for each muscle with increasing age. Between 4 and 12 mo
of
age, AR mRNA
levels increased (P < .05) in SP but remained unchanged in the ST
and
TB. Feeding regimen had no effect on muscle AR expression, but steers exhibiting
compensatory growth had higher AR mRNA levels than AL steers (P
<
.01) or bulls (P < .01). Our results show that AR expression is
muscle-specific and
may
be modulated by circulating testicular hormones. These data suggest
that
the regulation
of AR expression may be linked to allometric muscle growth patterns in
cattle and compensatory
gain in steers.
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