Various
external effects on PCR amplification
effiency
Quantification
of Insulin-like Growth Factor-1 (IGF-1) mRNA:
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)
Exp Clin Endocrinol
Diabetes. 1998;106(6): 506-513.
M. Pfaffl; F.
Schwarz & H. Sauerwein (1998)
Quantification of
the insulin
like growth factor-1 (IGF-1) mRNA: Modulation of growth intensity
by
feeding results in
inter- and intra-tissue specific differences of IGF-1 mRNA expression
in steers
Experimental and
Clinical Endocrinology & Diabetes 106: 513-520.
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
standardiser qRT-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 concentrations as 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.
Introduction
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.
Results
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 toPCR 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.
Sensitivity
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 androgen receptor mRNA in tissues by competitive
co-amplification
of a template in reverse
transcription-polymerase chain reaction.
Malucelli A,
Sauerwein H, Pfaffl MW, Meyer HHD
J Steroid Biochem
Mol Biol.
1996 Aug;58(5-6): 563-568.
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.
Effect
of DNA damage
on PCR amplification efficiency with the relative threshold cycle
method.
Sikorsky JA, Primerano DA, Fenger TW, Denvir J.
Biochem Biophys Res Commun. 2004 Oct 22;323(3): 823-8930
Department of Microbiology, Immunology and Molecular Genetics, Joan C.
Edwards
School of Medicine, Marshall University, Huntington, WV 25704, USA
Polymerase stop
assays used to
quantify DNA damage assume that single lesions are sufficient to
block polymerase
progression. To test the effect of specific lesions on PCR
amplification
efficiency, we amplified synthetic 90 base oligonucleotides containing
normal
or modified DNA bases using real-time PCR and determined the relative
threshold
cycle amplification efficiency of each template. We found that while
the
amplification efficiencies of templates containing a single
8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were not significantly
perturbed, the
presence of a single 8-oxo-7,8-dihydro-2'-deoxyadenosine,
abasic site, or a cis-syn thymidine dimer dramatically reduced
amplification
efficiency. In addition, while templates containing two 8-oxodGs
separated
by 13 bases amplified as well as the unmodified template, the presence
of two tandem 8-oxodGs substantially hindered amplification. From these
findings, we
conclude that the reduction in polymerase progression is dependent on
the
type of damage and the relative position of lesions within the template.
Amplification
effiency of thermostable DNA polymerases
Bahram Arezi,
Weimei Xing,
Joseph A. Sorge, and Holly H. Hogrefe*
Stratagene Cloning Systems, 11011 North Torrey Pines Road, La Jolla, CA
92037, USA
Analytical Biochemistry 321 (2003) 226–235

The
amplification efficiencies of
several polymerase chain reaction (PCR) enzymes were compared using
real-time quantitative PCR with SYBR Green I detection.
Amplification data collected during the exponential phase of PCR are
highly reproducible, and PCR enzyme performance
comparisons
based upon efficiency measurements are considerably more accurate than
those based on endpoint analysis. DNA polymerase
efficiencies were determined under identical conditions using five
different amplicon templates that varied in length or
percentage GC
content. Pfu- and Taq-based formulations showed similar efficiencies when
amplifying shorter targets (<900 bp) with
45 to 56% GC content. However, when amplicon length or GC content was
increased, Pfu formulations with dUTPase exhibited
significantly higher
efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss
the
implications of these results.
Evaluation
of
Real-Time PCR Amplification Efficiencies to Detect PCR Inhibitors
Elias J. Kontanis and Floyd A. Reed.
J Forensic Sci, July 2006, Vol. 51, No. 4

Real-time
PCR
analysis is a sensitive template DNA quantitation strategy that has
recently gained considerable attention in the forensic
community. However, the utility of real-time PCR methods extends beyond
quantitation and allows for simultaneous evaluation of template
DNA extraction quality. This study presents a computational method that
allows analysts to identify problematic samples with statistical
reliability
by comparing the amplification efficiencies of unknown template DNA
samples with clean standards. In this study, assays with varying
concentrations
of tannic acid are used to evaluate and adjust sample-specific
amplification efficiency calculation methods in order to optimize their
inhibitor
detection capabilities. Kinetic outlier detection and prediction
boundaries are calculated to identify amplification efficiency
outliers. Sample-specific
amplification efficiencies calculated over a four-cycle interval
starting at the threshold cycle can be used to detect reliably the
presence
of 0.4 ng of tannic acid in a 25 mL PCR reaction. This approach
provides analysts with a precise measure of inhibition severity when
template
samples are compromised. Early detection of problematic samples allows
analysts the opportunity to consider inhibitor mitigation strategies
prior to genotype or DNA sequence analysis, thereby facilitating sample
processing in high-throughput forensic operations.
Influence of
segmenting fluids on efficiency, crossing point and fluorescence level
in real time quantitative PCR.
E.J. Walsh
· C. King · R. Grimes · A. Gonzalez
Biomed
Microdevices (2006) 8: 59–64

The
two-phase segmented flow approach to the processing and quantitative
analysis of biological samples in microdevices offers significant
advantages over the singlephase continuous flow methodology. Despite
this, little is known about the compatibility of samples and reactants
with segmenting fluids, although a number of investigators have
reported reduced yield and inhibition of enzymatic reactions depending
on the segmenting fluid employed. The current study addresses the
compatibility of various segmenting fluids with real time
quantitativePCRto understand the physicochemical requirements of this
important reaction in biotechnology. The results demonstrate that
creating a static segmenting fluid/PCR mix interface has a negligible
impact on the reaction efficiency, crossing threshold and end
fluorescence levels using a variety of segmenting fluids. The
implication is then that the previously reported inhibitory effects are
the result of the dynamic motion between the segmenting fluid and the
sample in continuously flowing systems. The results presented here are
a first step towards understanding the limitations of the segmented
flow methodology, which are necessary to bring this approach into
mainstream
Quantitative
real-time polymerase chain reaction: methodical analysis and
mathematical model.
Wilkening
S, Bader A.
J
Biomol Tech. 2004 Jun;15(2):107-11.
German
Research Centre for Biotechnology, Braunschweig, Germany.

Real-time
polymerase chain reaction was established for 16 genes using the
LightCycler system to
evaluate gene expression in human hepatocytes. During the experiments a
large
set of data has been obtained. These data have now been evaluated with
respect to template stability, accuracy of melting curve analysis, and
reproducibility. In addition, the statistical evaluation of the
efficiencies
of all 16 polymerase chain reactions led to a new mathematical model.
To examine
template stability, the degradation of mRNA and cDNA was determined at
different temperatures. Surprisingly, cDNA, which was obtained by
first-strand
synthesis, appeared to degrade significantly faster than the respective
mRNA.
Melting curve analysis is a fast and sensitive method to check for
polymerase chain
reaction specificity. However, we show that two transcription
variants of the glutathione S-transferase 1 gene, with over 100 bp
length difference,
could not be distinguished by this method. Furthermore, an equation
was set up describing the correlation between polymerase chain reaction
efficiency
and crossing point. This equation can be used to estimate the number of
template molecules
without having a standard of known concentration. Finally, experimental
reproducibility of the real-time polymerase chain reaction was defined.
Enhancement in the
efficiency of polymerase chain reaction by TiO2 nanoparticles:
crucial
role of enhanced thermal conductivity.
Abdul Khaliq R, Sonawane PJ, Sasi BK, Sahu BS, Pradeep T,
Das SK, Mahapatra NR.
Nanotechnology. 2010 Jun 25;21(25):255704. Epub 2010 Jun 2.
Department of Biotechnology, Indian Institute of
Technology Madras, Chennai, India.

Improvement of the
specificity and efficiency of the polymerase chain reaction (PCR) by
nanoparticles is an emerging area of research. We observed that TiO(2)
nanoparticles of approximately 25 nm diameter caused significant
enhancement of PCR efficiency for various types of templates (namely
plasmid DNA, genomic DNA and complementary DNA). By a series of
experiments, the optimal TiO(2) concentration was determined to be 0.4
nM, which resulted in up to a seven-fold increase in the amount of PCR
product. As much as 50% reduction in overall reaction time (by
reduction of the number of cycles and the time periods of cycles) was
also achieved by utilizing TiO(2) nanoparticles without compromising
the PCR yield. Investigations of the mechanism of such PCR enhancement
by simulations using the 'Fluent K epsilon turbulent model' provided
evidence of faster heat transfer in the presence of TiO(2)
nanoparticles. Consistent with these findings, TiO(2) nanoparticles
were observed to augment the denaturation of genomic DNA, indicating
more efficient thermal conductivity through the reaction buffer. TiO(2)
nanoparticle-assisted PCR may be useful for profound reduction of the
overall PCR reaction period and for enhanced amplification of DNA
amplicons from a variety of samples, including GC-rich templates that
are often observed to yield unsatisfactory results.
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