Determination of PCR Efficiency (3) Determination of PCR Efficiency (main) Determination of PCR Efficiency (1) Determination of PCR Efficiency (2) Determination of PCR Efficiency (4) Determination of PCR Efficiency (5) New papers around PCR efficiency estimation and correction - PCR inhibition by
reverse transcriptase leads to an overestimation of amplification
efficiency.
Suslov O, Steindler DA.**download PDF**
- Overview article - Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction Sheng Zhao and Russell D. Fernald
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PDF
- Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis. Cikos S, Bukovská A, Koppel J., 2007 download PDF
- Enhancing the
efficiency of a PCR using gold nanoparticles.
Li M, Lin YC, Wu CC, Liu HS. 2006 download PDF
- Technical
Note - Evaluation
of Real-Time PCR
Amplification Efficiencies to Detect PCR Inhibitors
Elias J. Kontanis and Floyd A. Reed, 2005**download PDF**
**Estimation of the reaction efﬁciency in polymerase chain reaction**Nadia Lalam, 2006
**download PDF**
- Molelling
the PCR amplification process by a size-dependent branching process and
estimation of the efficiency
Lalam et al., 2004**download PDF**
- Statistical Inference for Quantitative Polymerase Chain Reaction Using a Hidden Markov Model: A Bayesian Approach Lalam, 2007 download PDF
- Evaluation
of absolute quantitation by nonlinear regression in probe-based
real-time PCR
Goll et al., 2006 download PDF
- Mathematical Model of Real-Time PCR Kinetics Gevertz et al., 2005 download PDF
- Quantitative
real-time RT-PCR based transcriptomics: Improvement of evaluation methods.
PhD. Thesis - Ales Tichopad, 2004 Lehrstuhl für Physiologie, Fakultät Wissenschaftszentrum Weihenstephan, Technische Universität München, Germany.
PCR inhibition by
reverse transcriptase leads to an overestimation of amplification
efficiency.
Suslov O, Steindler
DA.
This study addresses
the problem of PCR inhibition by reverse transcriptase. It has
been shown that
the inhibition occurs mostly when a small amount of RNA is taken
for RT
reaction, and it is more visible for rarely expressed transcripts. We
show here that the
inhibition takes place regardless of what amount of template
is utilized
for RT. The inhibition possesses a global nature, i.e. the amplification
of any
given transcript may be compromised with different levels of inhibition.
The
process of inhibition also explains wrongfully derived PCR amplification
efficiencies, sometimes more than 100%, when the sequential dilutions
of
unpurified RT sample are utilized to build the calibration curve. The
RT influences PCR
not only by inhibiting it. When microgram(s) of RNA are taken
for RT
reaction, reverse transcriptase may cause overamplification of some transcripts
under
certain PCR conditions. The possible mechanism of RT influence on
PCR is presented,
and a purification method is implemented to remove the effects
of RT on PCR.McKnight Brain Institute of the University of Florida, FL, USA. Nucleic Acids Res. 2005 Nov 27;33(20):e181. Absolute and
relative
real-time PCR in the quantification of tst gene expression among
methicillin-resistant Staphylococcus aureus: evaluation by two
mathematical models.
AIM:
Absolute and relative quantitative real-time reverse transcriptase
polymerase chain reaction (RT-PCR) by the use of two mathematical
models were applied in order to study the expression of tst gene
encoding the toxic shock syndrome toxin-1 (TSST-1), among
methicillin-resistant Staphylococcus aureus (MRSA). Chini V, Foka A, Dimitracopoulos G, Spiliopoulou I. Lett Appl Microbiol. 2007 Nov;45(5):479-84. Department of Microbiology, School of Medicine, University of Patras, Patras, Greece. METHODS AND RESULTS: Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods. CONCLUSIONS: tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.
In real-time PCR data
analysis, the cycle threshold (CT) method is currently the gold
standard. This method is based on an assumption of equal PCR efficiency
in all reactions, and precision may suffer if this condition is not
met. Nonlinear regressionanalysis (NLR) or curve fitting has therefore
been suggested as an alternative to the cycle threshold method for
absolute quantitation. The advantages of NLR are that the individual
sample efficiency is simulated by the model and that absolute
quantitation is possible without a standard curve, releasing reaction
wells for unknown samples. However, the calculation method has not been
evaluated systematically and has not previously been applied to a
TaqMan platform. Aim: To develop and evaluate an automated NLR
algorithm capable of generating batch production regression
analysis.Total RNA samples extracted from human gastric mucosa were
reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan
real-time PCR. Fluorescence data were analysed by the regular CT method
with a standard curve, and by NLR with a positive control for
conversion of fluorescence intensity to copy number, and for this
purpose an automated algorithm was written in SPSS syntax. Eleven
separate regression models were tested, and the output data was
subjected to Altman-Bland analysis. The
Altman-Bland analysis showed that the best regression model yielded
quantitative data with an intra-assay variation of 58% vs. 24% for the
CT derived copy numbers, and with a mean inter-method deviation of
x0.8. NLR
can be automated for batch production analysis, but the CT method is
more precise for absolute quantitation in the present setting. The
observed inter-method deviation is an indication that assessment of the
fluorescence conversion factor used in the regression method can be
improved. However, the versatility depends on the level of precision
required, and in some settings the increased cost effectiveness of NLR
may justify the lower precision.
Quantitative
real-time RT-PCR based transcriptomics: Improvement of
evaluation methods.
PhD. Thesis - Ales Tichopad Lehrstuhl für Physiologie, Fakultät Wissenschaftszentrum Weihenstephan, Technische Universität München, Germany. SUMMARY
Quantitative real-time polymerase chain reaction (qRT-PCR) is a new method for reliable quantification of low-abundance mRNA in biological samples. Since the strength of the fluorescence signal emitted by the report dye should be proportional to the produced DNA amount, the fluorescence monitoring enables visualisation of the full reaction trajectory. The reaction trajectory can be then extrapolated back to an input concentration. RNA extraction can introduce unwanted contaminants into the sample, inhibiting the reverse transcription (RT) as well as the PCR reaction. These inhibitions cause then the reaction to precede sample-specific. In addition, the amplification efficiency varies not only between samples, but also along the recorded amplification trajectory of a single sample. Consequently, a correct determination of each probe’s PCR efficiency as well as a good standardization of the raw expression estimators is of great importance for a correct interpretation of results. To find a solution to above problems a series of biological experiments with RNA extracted from various ovine and bovine tissues and from cultured leukocytes was carried out. Constant amount of RNA was then reverse–transcribed to cDNA. All PCR runs were performed on a LightCycler instrument and Fluorescence data was saved in the LightCycler software. Based on this data, mathematical models together with statistical procedures were developed and validated. These can investigate the optimal quantification range and exactly determine its real-time PCR efficiency. Additionally, methods were developed to disclose heterogeneity between probes. All these procedures contribute to better quality of results obtained. Resulting from these standardisations, a decision algorithm for a proper analysis of the qRT-PCR data was designed. |