Real-Time PCR: A
Review of Approaches to Data Analysis.
V. Rebrikov and D. Yu. Trofimov
JSC, Moscow, 115478 Russia
Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 5, pp.
registration of the accumulation of polymerase chain reaction (PCR)
products in the course of amplification
(real-time PCR) requires specific equipment, i.e., detecting amplifiers
capable of recording the level of fluorescence
in the reaction tube during amplicon formation. When the time of the
reaction is complete, researchers are able
to obtain DNA accumulation graphs. This review discusses the most
promising algorithms of the analysis of
real-time PCR curves and possible errors, caused by the software used
or by operators' mistakes. The data included
will assist researchers in understanding the features of a method to
obtain more reliable results.
Calculator to convert
the slope produced by a QPCR standard curve to % efficiency. It also
gives the exponent and amplification. This
calculator uses the slope produced by a QPCR standard curve to
calculate the efficiency of the PCR reaction. Slopes between -3.1 and
-3.6 giving reaction efficiencies between 90 and 110% are typically
formula for this
calculation is Efficiency
= -1 + 10(-1/slope)
dilution curve and slope calculation"
E = 10^[–1/slope]
Efficiency of PCR
Note #10 by Stratagene
on the LightCycler
Rasmussen, R (2001)
In: Meuer, S,
Nakagawara, K, eds.
Real-time PCR, Methods and Applications Springer Press, Heidelberg;
Quantification of purified PCR product of the human
HER2/neu gene in the LightCycler Instrument. The reaction
was monitored with SYBR Green I. Acquisitions were taken once
per cycle after extension.
2: A standard curve constructed from the data in figure 1. The
slope of the line is -1/log (efficiency) giving an efficiency in this
case of 1.73. The intercept is the log of the amount of DNA at
threshold divided by the log of the efficiency.
quantitative real-time PCR method for detection of B-Lymphocyte
by comparison of
kappa and lambda Immunoglobulin Light Chain Expression.
Ståhlberg, Pierre Åman, Börje Ridell, Petter Mostad,
and Mikael Kubista (2003)
Chemistry 49(1): 51-59
new mathematical model for relative quantification in real-time RT-PCR.
W. Pfaffl (2001)
Nucleic Acids Res.
2001 May 1; 29(9): E45-E45.
validation of novel and conventional approaches
to quantitative real-time PCR data analysis.
Stuart N. Peirson, Jason N. Butler
and Russell G. Foster (2003)
PCR is being used increasingly as the method of choice for mRNA
quantification, allowing rapid analysis of gene
expression from low quantities of starting template. Despite
a wide range of approaches, the same principles underlie all data
analysis, with standard approaches broadly classiffed as
either absolute or relative. In this study we use a variety of absolute
and relative approaches of data analysis to investigate nocturnal c-fos
expression in wild-type and retinally degenerate mice.
In addition, we apply a simple algorithm to calculate the amplifcation
effciency of every sample from its amplifcation profle. We confrm that
nocturnal c-fos expression in the rodent eye originates from the
photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos
expression in mice lacking rods and cones. Furthermore,
illustrate that differences in the results obtained from absolute and
relative approaches are underpinned by differences in the calculated
PCR effciency. By calculating the amplifcation effciency from the
samples under analysis, comparable results
may be obtained without the need for standard curves. We have automated
this method to provide a means of streamlining the real-time PCR
process, enabling analysis of experimental samples based upon their own
reaction kinetics rather than those of artificial standards.
quantitative kinetic PCR and the application of standard curves.
provides a simple means of analysing real-time PCR data from raw
flurescence data. This allows an automatic calculation of amplification
kinetics, as well as performing the subsequent calculations for
relative quantification and calculation of assay
Amplification efficiencies are also tested to dtect anomalus samples
within groups (outlayers) and differences between experimatal groups
Rutledge RG, Cote C.
Acids Res. 2003 Aug 15;31(16):e93
Resources Canada, Canadian Forest Service, Laurentian Forestry Centre,
1055 du P.E.P.S., PO Box 3800, Sainte-Foy, Quebec G1V 4C7, Canada
monitoring of DNA
amplification is the basis of real-time PCR, from which target
concentration can be determined from the fractional cycle at which a
threshold amount of
amplicon DNA is produced. Absolute quantification can be achieved using
curve constructed by amplifying known amounts of target DNA. In this
mathematics of quantitative PCR are examined in detail, from which
fundamental aspects of the threshold method and the application of
are illustrated. The construction of five replicate standard curves for
pairs of nested primers was used to examine the reproducibility and
quantitative variation using SYBER Green I fluorescence. Based upon
analysis the application of a single, well-constructed standard curve
provide an estimated precision of +/-6-21%, depending on the number of
required to reach threshold. A simplified method for absolute
is also proposed, in which quantitative scale s determined
by DNA mass at
statistically verified quantification of relative mRNA abundances using SYBR Green I
and real-time RT-PCR.
Marino JH, Cook P, Miller KS.
J Immunol Methods. 2003 Dec;283(1-2):291-306.
Faculty of Biological Sciences, The University of Tulsa,
600 S. College Avenue, Tulsa, OK 74104-3189, USA.
the many methods currently available for quantifying mRNA transcript abundance,
transcription- polymerase chain reaction (RT-PCR) has proved to
be the most
sensitive. Recently, several protocols for real-time relative RT-PCR
using the reporter dye SYBR Green I have appeared in the literature. In
sample and control mRNA abundance is quantified relative to an internal
reference RNA whose abundance
is known not to change under the differing experimental
conditions. We have developed new data analysis procedures
for the two most
promising of these methodologies and generated data appropriate
to assess both
the accuracy and precision of the two protocols. We demonstrate
that while both
methods produce results that are precise when 18S rRNA
is used as an internal
reference, only one of these methods produces consistently
results. We have
used this latter system to show that mRNA abundances can
be accurately measured
and strongly correlate with cell surface protein and
expression as assessed by flow cytometry under different
conditions of B
Impact of DNA
polymerases and their buffer systems on quantitative real-time PCR.
Wolffs P, Grage H,
Hagberg O, Radstrom P.
J Clin Microbiol.
Lund Institute of Technology, Mathematical Statistics, Lund University,
SE-221 00 Lund, Sweden.
of five DNA polymerase-buffer systems on real-time PCR showed
choice of both DNA polymerase and the buffer system affected the
efficiency as well as the detection window. The analytical
for different systems changed clearly, leading us to conclude that
quantitative measurements on single-data-set standard curves can lead
fluorogenic real-time qPCR inhibition using the novel custom Excel file
system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently
high fidelity qPCR reactions.
Gallup and Mark R. Ackermann
of Veterinary Pathology, College of Veterinary Medicine, Iowa State
University. Ames, Iowa 50011-1250. USA.
Proced. Online 2006;8:87-152.
purpose of this manuscript is to discuss fluorogenic real-time
quantitative polymerase chain reaction (qPCR) inhibition and to
introduce/define a novel Microsoft Excel-based file system which
provides a way to detect and avoid inhibition, and enables
investigators to consistently design dynamically-sound, truly
LOG-linear qPCR reactions very quickly. The qPCR problems this
invention solves are universal to all qPCR reactions, and it performs
all necessary qPCR set-up calculations in about 52 seconds (using a
pentium 4 processor) for up to seven qPCR targets and seventy-two
samples at a time – calculations that commonly take capable
investigators days to finish. We have named this custom Excel-based
file system "FocusField2- 6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR
set-up tool), and are in the process of transforming it into
professional qPCR set-up software to be made available in 2007. The
current prototype is already fully functional.
of DNA damage on PCR amplification efficiency with the relative
JA, Primerano DA, Fenger TW, Denvir J.
Biophys Res Commun. 2004 Oct 22;323(3):823-30.
of Microbiology, Immunology and Molecular Genetics, Joan C. Edwards
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.
E-Method: a highly accurate technique for gene-expression analysis
Nature, Application Note, July 2006
Roche Applied Science
has repeatedly set standards for high-speed real-time PCR systems. The
System offers different methods of data analysis for relative
quantification of geneexpressionbehavior. Whereas the
∆∆CT Method provides fast, easy analysis of gene expression, the
Roche Applied Science can produce more accurate relative quantification
data bycompensating for
differences in target and reference-gene amplification efficiency,
either within anexperiment or between
480 System - Innovative solutions for relative quantification
Estimation via increase
in "absolute fluorescence" method 1
and validation of an externally standardised quantitative Insulin
growth factor-1 (IGF-1) RT-PCR
using LightCycler SYBR ® Green I technology.
Pfaffl, MW (2001)
In: Meuer, S,
Nakagawara, K, eds. Rapid Cycle Real-time PCR, Methods and Applications
Heidelberg, ISBN 3-540-66736-9
via increase in "absolute
E = ( Rn
B / Rn A ) ^ [ 1 / CP B - CP A ]
quantitative method of real time RT-PCR
assay based on simulation of
polymerase chain reaction kinetics.
Liu W & Saint
2002 302(1): 52-59.
Please note that a
typographical error occurred in equation
in this paper.
Here is the correct
equation for calculating efficiency, and
via increase in "absolute
3 (3 data points)
estimations of PCR amplification rates
& Jacob C. (1998)
Quantification (eds. Francois Ferre)
applications of the Polymerase Chain Reaction (PCR), also known as
Quantitative-PCR (Q-PCR) are intended either to determine the number of
copies of a given
nucleic acid sequence, or more generally, to determine the relative abundance
of two sequences. Current methods to determine exact numbers of molecules overcome
the determination of the amplification rate by assuming
amplification rates for a target DNA sequence and a standard of known quantity
introduced into the experiment design, so that only the ratio of amplified
products need be determined. Violations of the hypothesis of identical
amplification rates for two sequences will result in a systematic
bias in the
experiment results that underestimates or overestimates the initial
Acquisition of kinetic PCR data was pioneered by Higuchi et al. (Higuchi et al.,
1993; Higuchi et al., 1992) and commercial instruments have been available
since early 1996. Kinetic data provide a new way to determine the
rate, and we can foresee that their availability will rekindle interest
in the algorithms
used to compute the initial quantities
of DNA sequences. Analysis of kinetic PCR
patterns will soon make its way into
the family of recipes that have been in use
for some years in this field. This chapter provides evidence that
a statistical analysis of the amplification rate is critical to ensuring
estimate of the initial copy number.
via increase in "absolute
analysis of quantitative real-time PCR data.
Ruijter JM, Deprez RH, Moorman AF. (2003)
2003 Mar 13;339(1): 62-66
Anatomy and Embryology K2-283, Experimental and Molecular Cardiology Group,
Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ,
Amsterdam, The Netherlands
Quantification of mRNAs
using real-time polymerase chain
reaction (PCR) by monitoring the product
formation with the fluorescent dye
SYBR Green I is being extensively used in
neurosciences, developmental biology,
and medical diagnostics.
Most PCR data analysis procedures assume that the PCR efficiency
for the amplicon
of interest is constant or even, in the case of the comparative C(t) method,
equal to 2. The latter method already leads to a 4-fold
error when the
PCR efficiencies vary over just a 0.04 range. PCR efficiencies of
usually calculated from standard curves based on either known RNA
inputs or on
series of a reference cDNA sample. In this paper we show
that the first
approach can lead to PCR efficiencies that vary over a 0.2 range,
second approach may be
off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per
cycle number data as an assumption-free method to calculate starting
concentrations of mRNAs and PCR efficiencies for each
computer program to perform this calculation is available on
direct download here
the effect of unequal PCR efficiencies on the result of the comparative
the linear regression calculations implemented in a Microsoft Excelw
spreadsheet for determining starting concentrations and PCR
efficiencies per sample.
A new reverse
reaction method for accurate quantification.
BMC Biotechnology (2003)
transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique
to measure and to
compare mRNA levels among samples. However, it is extremely difficult
linearity across the entire procedure, especially at the step of PCR
genes have been used as baseline controls to be co-amplified with target
normalize the amplification efficiency, but development or selection of
reliable controls itself has created a new challenge.
Here, we describe a new quantitative RT-PCR to compare two mRNA samples
directly without the requirement of
control DNAs for reference. First, chimeric RT primers carrying
and universal PCR priming sequences with or without a linker for size
were utilized to generate cDNAs. The size-different cDNAs were then
combined in a single reaction for PCR
amplification using the same primer set. The two amplified products were
detected with gel electrophoresis and fluorescence imaging. Relative
abundance of the two products was obtained after a
Conclusion: This methodology is simple and
accurate as indicated
by equal amplification efficiency throughout PCR
cycling. It is also
easily implemented for many existing
protocols. In addition, parameters
affecting RT linearity are characterized in this report.
Q-Gene: processing quantitative
of the first PCR cycles in real-time comparative gene quantifications.
Nogva HK, Rudi K.
Biotechniques. 2004 Aug;37(2):246-8, 250-3.
Norwegian Food Research Institute, AS, Norway.
There is an
in real-time PCR that the amplification efficiency is equal from the
cycles until a signal
can be detected. In this
study, we evaluated this
assumption by analyzing genes with known gene copy number using
comparative gene quantifications. Listeria monocytogenes has six 23S
and one copy of the hlyA gene. We determined 23S rRNA gene copy
numbers between 0.9 and 1.6 relative to hlyA when applying the
quantification approach. This paper focuses on the first cycles of PCR
to explain the
difference between known and determined gene copy numbers. Both
experimental evaluations were done. There are three different products
1-3) dominating in the first cycles. Type 1 is the original target,
type 2 are
undefined long products, while type 3 are products that accumulate
PCR. We evaluated the effects of type 1 and 2 products during the first
the target DNA with a restriction enzyme that cuts outside the
boundaries of the PCR products. The digestion resulted in a presumed
amplification efficiency for type 1 and 2 products. Differences in the
amplification efficiencies between type 1, 2, and 3 products may
explain part of the
error in the gene copy number determinations using real-time PCR
gene quantifications. Future applications of real-time PCR
should account for the effect of the first few PCR cycles on the
in the Cq value observed with hydrolysis probe based quantitative PCR
can be corrected with the estimated PCR efficiency value.
Tuomi JM, Voorbraak F, Jones DL, Ruijter JM.
Methods. 2010 Apr;50(4): 313-22
For real-time monitoring of PCR amplification of DNA,
(qPCR) assays use various fluorescent reporters. DNA binding molecules
and hybridization reporters (primers and probes) only fluoresce when
bound to DNA and result in the non-cumulative increase in observed
fluorescence. Hydrolysis reporters (TaqMan probes and QZyme primers)
become fluorescent during DNA elongation and the released fluorophore
remains fluorescent during further cycles; this results in a cumulative
increase in observed fluorescence. Although the quantification
threshold is reached at a lower number of cycles when fluorescence
accumulates, in qPCR analysis no distinction is made between the two
types of data sets. Mathematical modeling shows that ignoring the
cumulative nature of the data leaves the estimated PCR efficiency
practically unaffected but will lead to at least one cycle
underestimation of the quantification cycle (C(q) value), corresponding
to a 2-fold overestimation of target quantity. The effect on the target
reference ratio depends on the PCR efficiency of the target and
reference amplicons. The leftward shift of the C(q) value is dependent
on the PCR efficiency and with sufficiently large C(q) values, this
shift is constant. This allows the C(q) to be corrected and unbiased
target quantities to be obtained.
new method for robust quantitative and qualitative analysis of
Eric B. Shain
John M. Clemens
Molecular Inc., 1300 E. Touhy Avenue, Des Plaines, IL 60018, USA
Acids Research, 2008, Vol. 36, No. 14 e91
automated data analysis method for real-time PCR needs to exhibit
robustness to the factors that routinely impact the measurement and
analysis of real-time PCR data. Robust analysis is paramount to
providing the same interpretation for results regardless of the skill
of the operator performing or reviewing the work. We present a new
method for analysis of real-time PCR data, the maxRatio method, which
identifies a consistent point within or very near the exponential
region of the PCR signal without requiring user intervention. Compared
to other analytical techniques that generate only a cycle number,
maxRatio generates several measurements of amplification including
cycle numbers and relative measures of amplification efficiency and
curve shape. By using these values, the maxRatio method can make highly
reliable reactive/nonreactive determination along with quantitative
evaluation. Application of the maxRatio method to the analysis of
quantitative and qualitative real-time PCR assays is shown along with
examples of method robustness to, and detection of, amplification
University Eye Hospital Tuebingen,
72076 Tuebingen, Germany
Q-Gene is an application
for the processing of
RT–PCR data. It offers the user the possibility to freely choose
between two principally different procedures to calculate normalized
gene expressions as either
means of Normalized Expressions or Mean Normalized Expressions. In this
contribution it will be shown that the calculation of Mean Normalized
has to be used for processing simplex PCR data, while multiplex PCR
should preferably be processed by calculating Normalized Expressions.
two procedures, which are currently in widespread use and regarded as
or less equivalent alternatives, should therefore specifically be
according to the quantification procedure used.
kinetic model of quantitative
real-time polymerase chain reaction.
Mehra S, Hu WS.
Chemical Engineering and Materials Science, University of Minnesota,
421 Washington Avenue SE, Minneapolis, MN 55455-0132, USA.
2005 Sep 30;91(7):848-60.
chain reaction (PCR) is one of the most sensitive and accurate methods for
quantifying transcript levels especially for those expressed at low abundance. The selective
amplification of target DNA over multiple cycles allows
concentration to be determined. The amplification rate is a complex interplay of the
operating conditions, initial reactant concentrations, and reaction rate constants.
Experimentally, the compounded effect of all factors is quantified in terms of an
effective efficiency, which is estimated by curve fitting to the amplification
data. We present a comprehensive model of PCR to study
the effect of various
reactant concentrations on the amplification efficiency.
The model is used
to calculate the kinetic progression of the target DNA concentration with cycle
number under conditions when different species are stoichiometrically or
kinetically limiting. The reaction efficiency remains constant for the initial
cycles. As the primer concentration becomes limiting, the efficiency is marked by a
gradual decrease. This is in contrast to a steep decline
limiting conditions. Under some conditions, commonly used experimentally, increasing
primer concentration has the adverse effect of reducing
the final amplified
template concentration. This phenomenon seen at times
explained by the simulation results under rate limiting enzyme concentrations. Primer
dimer formation is shown to significantly affect the
reaction rates, effective
efficiency, and the estimated initial concentrations.
This model, by
describing the interplay of the many operating variables,
will be a useful
tool in designing PCR conditions and evaluating its results.
Outlier Detection (KOD) in
Tzachi Bar, Anders Stahlberg, Anders Muszta and Mikael Kubista
NAR Vol 31 (17): e105
Chemistry and Bioscience, Chalmers University of Technology,
405 30 Gothenburg,
Sweden, 2Department of Mathematical Statistics, Eklandagatan 86, 412
Sweden and 3TATAA
Biocenter, Medicinargatan 7B, 405 30 Gothenburg, Sweden
Real-time PCR is becoming the
method of choice for precise quantification of minute amounts of
nucleic acids. For proper comparison of samples, almost all
quantification methods assume similar PCR effciencies in the
exponential phase of the reaction. However, inhibition of PCR is common
when working with biological samples and may invalidate the assumed
similarity of PCR effiencies. Here we present a statistical method,
Kinetic Outlier Detection (KOD), to detect samples with dissimilar
effiiencies. KOD is based on a comparison of PCR effciency, estimated
from the amplifiation curve of a test sample, with the mean PCR
effiency of samples in a training set. KOD is demonstrated and
validated on samples with the same initial number of template
molecules, where PCR is inhibited to various degrees by elevated
concentrations of dNTP; and in detection of cDNA samples with an
aberrant ratio of two genes. Translating the dissimilarity in
efficiency to quantity, KOD identifies outliers that differ by
1.3±1.9-fold in their quantity from normal samples with a
P-value of 0.05. This precision is higher than the minimal 2-fold
difference in number of DNA molecules that real-time PCR usually aims
to detect. Thus, KOD may be a useful tool for outlier detection in
assessment for relative quantification by real-time PCR.
T, Muszta A.
2005 Sep;39(3): 333-338
University of Technology, Gothenburg, Sweden.
For proper relative quantification by real-time PCR, compared samples
similar PCR efficiencies. To test this prerequisite, we developed two
quality tests: (i)
adjustment of a test for kinetic outlier detection (KOD) to relative
quantification; and (ii) comparison of the efficiency variance of test
samples with the
efficiency variance of samples with highly reproducible quantification.
tests were applied on relative quantification of two genes in 30 sets
replicate samples (same treatment, different animals). Ten low-quality
28 outliers were identified. The low-quality sets showed higher
variation (cv)% of DNA quantities in replicate experiments than
sets (63% versus 26%; P = 0.001) and contained a higher proportion of
outlying quantities (35% versus 5.9%; P = 0.001) when individual
samples were detected
by adjusted KOD. Outlier detection with adjusted KOD reduced the false
detection of outliers by 2/3 compared with the previous, nonadjusted
of KOD (20% versus 5.9%; P = 0.001). We conclude that the presented
be used to assign technical reasons to outlying observations.
Quality control for quantitative PCR based on
amplification compatibility test.
Tichopad A, Bar T, Pecen L, Kitchen RR, Kubista M,
Methods. 2010 50(4): 308-312
Quantitative qPCR is a
routinely used method for the accurate quantification of nucleic acids.
Yet it may generate erroneous results if the amplification process is
obscured by inhibition or generation of aberrant side-products such as
primer dimers. Several methods have been established to control for
pre-processing performance that rely on the introduction of a
co-amplified reference sequence, however there is currently no method
to allow for reliable control of the amplification process without
directly modifying the sample mix. Herein we present a statistical
approach based on multivariate analysis of the amplification response
data generated in real-time. The amplification trajectory in its most
resolved and dynamic phase is fitted with a suitable model. Two
parameters of this model, related to amplification efficiency, are then
used for calculation of the Z-score statistics. Each studied sample is
compared to a predefined reference set of reactions, typically
calibration reactions. A probabilistic decision for each individual
Z-score is then used to identify the majority of inhibited reactions in
our experiments. We compare this approach to univariate methods using
only the sample specific amplification efficiency as reporter of the
compatibility. We demonstrate improved identification performance using
the multivariate approach compared to the univariate approach. Finally
we stress that the performance of the amplification compatibility test
as a quality control procedure depends on the quality of the reference
Efficiency clustering for low-density microarrays and
its application to qPCR
Eric F Lock, Ryan Ziemiecke, J. S. Marron and Dirk P
BMC Bioinformatics 2010, 11
low-density arrays are used more and more frequently in biomedical
research, particularly those arrays that are based on quantitative
real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or
probes, and they bring with it the promise of being able to reliably
measure differences in target levels without the need to establish
absolute standard curves for each and every target. To achieve reliable
quantification all primer pairs or array probes must perform with the
primer-pairs differ significantly both in reliability and efficiency.
They can only be used in an array format if the raw data (so called CT
values for real-time QPCR) are transformed to take these differences
into account. We developed a novel method to obtain efficiency-adjusted
CT values. We introduce transformed confidence intervals as a novel
measure to identify unreliable primers. We introduce a robust
clustering algorithm to combine efficiencies of groups of probes, and
our results indicate that using n < 10 cluster-based mean
efficiencies is comparable to using individually determined efficiency
adjustments for each primer pair (N = 96-1024).
Careful estimation of
efficiency is necessary to avoid significant measurement inaccuracies.
Transformed confidence intervals are a novel method to assess and
interprete the reliability of an efficiency estimate in a high
throughput format. Efficiency clustering as developed here serves as a
compromise between the imprecision in assuming uniform efficiency, and
the computational complexity and danger of over-fitting when using
individually determined efficiencies.
Shape based kinetic outlier detection in real-time
Sisti D, Guescini M, Rocchi MB, Tibollo P, D'Atri M,
BMC Bioinformatics. 2010 12;11: 186
BACKGROUND - Real-time
PCR has recently become the technique of choice for absolute
and relative nucleic acid quantification. The gold standard
quantification method in real-time PCR assumes that the compared
samples have similar PCR efficiency. However, many factors present in
biological samples affect PCR kinetic, confounding quantification
analysis. In this work we propose a new strategy to detect outlier
samples, called SOD.
RESULTS - Richards
function was fitted on fluorescence readings to parameterize
the amplification curves. There was not a significant correlation
between calculated amplification parameters (plateau, slope and
y-coordinate of the inflection point) and the Log of input DNA
demonstrating that this approach can be used to achieve a "fingerprint"
for each amplification curve. To identify the outlier runs, the
calculated parameters of each unknown sample were compared to those of
the standard samples. When a significant underestimation of starting
DNA molecules was found, due to the presence of biological inhibitors
such as tannic acid, IgG or quercitin, SOD efficiently marked these
amplification profiles as outliers. SOD was subsequently compared with
KOD, the current approach based on PCR efficiency estimation. The data
obtained showed that SOD was more sensitive than KOD, whereas SOD and
KOD were equally specific.
CONCLUSION - Our
results demonstrated, for the first time, that outlier detection
can be based on amplification shape instead of PCR efficiency. SOD
represents an improvement in real-time PCR analysis because it
decreases the variance of data thus increasing the reliability of
INTERFACE - Cy0 is a new method in Real-time PCR analysis that does not
require the assumption of equal efficiency between unknowns and
standard curve (Michele Guescini, Davide Sisti, & Renato