qPCR NEWS - December 2011 - focus on qPCR efficiency

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Tue Dec 20 11:12:23 EST 2011


qPCR NEWS - December 2011 - focus on qPCR efficiency
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Dear researcher,
dear Gene Quantification page reader,

Our newsletter informs about the latest news in quantitative real-time
PCR (qPCR and RT-qPCR), which are compiled and summarised on the
Gene Quantification homepage. The focus of this newsletter issue is:

* qPCR efficiency calculation  -  sub-domain updated  -
http://Efficiency.gene-quantification.info
* real-time PCR Cycler  -  sub-domain updated  -  http://CYCLERS.gene-quant=
ification.info
* GenEx version 5  - download a free trial version  -
http://GenEx.gene-quantification.info
* MIQE qPCR APP for IOS and Android is available - http://MIQE.gene-quantif=
ication.info

If this newsletter is not displayed correctly by your email client,
please use following Link
http://qPCRnews.gene-quantification.info

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Determination of real-time PCR amplification efficiency

Individual samples generate different and individual fluorescence
histories in real-time PCR. The shapes of amplification curves differ
in the steepness of any fluorescence increase and in the absolute
fluorescence levels at plateau depending on background fluorescence
levels. The PCR efficiency has 'the 'major impact' on the fluorescence
history and the accuracy of the calculated expression result and is
critically influenced by PCR reaction components. The efficiency
evaluation is an essential marker in gene quantification procedure and
one of the important characteristicum of the MIQE guidelines. Constant
amplification efficiency in all compared samples is one important
criterion for reliable comparison between samples. This becomes
crucially important when analyzing the relationship between an unknown
sequence versus a standard sequence, which is performed in all
relative quantification models. In experimental designs employing
standardization with reference genes, the demand for invariable
amplification efficiency between target and standard is often ignored,
despite the fact that corrections have been suggested. A correction
for efficiency, as performed in efficiency corrected mathematically
models, is strongly recommended and results in a more reliable
estimation of the =91real expression ratio=92 compared to NO efficiency
correction. Small efficiency differences between target and reference
gene generate false expression ratio, and the researcher over- or
under-estimates the =91real=92 initial mRNA amount.

Find the latest literature about real-time PCR efficiency
determination:

Efficiency of the Polymerase Chain Reaction.
Booth CS, Pienaar E, Termaat JR, Whitney SE, Louw TM, Viljoen HJ.
Chem Eng Sci. 2010 65(17): 4996-5006.

pcrEfficiency: a Web tool for PCR amplification efficiency prediction.
Mallona I, Weiss J, Marcos EC.
BMC Bioinformatics. 2011 12: 404

Experimental Validation of a Fundamental Model for PCR Efficiency.
Louw TM, Booth CS, Pienaar E, Termaat JR, Whitney SE, Viljoen HJ.
Chem Eng Sci. 2011 Apr 15;66(8): 1783-1789.

Enhanced analysis of real-time PCR data by using a variable efficiency
model: FPK-PCR.
Lievens A, Van Aelst S, Van den Bulcke M, Goetghebeur E.
Nucleic Acids Res. 2011 Nov 18.

Validation of kinetics similarity in qPCR.
Bar T, Kubista M, Tichopad A.
Nucleic Acids Res. 2011 Oct 19.

A mechanistic model of PCR for accurate quantification of quantitative
PCR data.
Boggy GJ, Woolf PJ.
Department of Chemical Engineering, University of Michigan, Ann Arbor,
Michigan, United States of America.
PLoS One. 2010 5(8): e12355.

Shape based kinetic outlier detection in real-time PCR.
Sisti D, Guescini M, Rocchi MB, Tibollo P, D'Atri M, Stocchi V.
BMC Bioinformatics. 2010 12;11: 186

Quality control for quantitative PCR based on amplification
compatibility test.
Tichopad A, Bar T, Pecen L, Kitchen RR, Kubista M, Pfaffl MW.
Methods. 2010 50(4): 308-312

Efficiency clustering for low-density microarrays and its application
to qPCR
Eric F Lock, Ryan Ziemiecke, J. S. Marron and Dirk P Dittmer
BMC Bioinformatics 2010, 11

Assessing the performance capabilities of LRE-based assays for
absolute quantitative real-time PCR.
Rutledge RG, Stewart D.
PLoS One. 2010 5(3): e9731.

Bias 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 50(4): 313-22

... ... and more =3D>  http://Efficiency.gene-quantification.info

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UPDATE - real-time PCR Cycler

On http://CYCLERS.gene-quantification.info the most prominent real-
time PCR cycler are described.
In the cycler descriptions the specifications and the advantages of
the displayed systems are shown. Which real-time platform meets your
requirements best, depends on your research application. Some of the
systems are designed for research with low capacities and others are
for high-throughput applications, most in combination with pipetting
robots. Most of them use solid-block for thermal cycling, other use
hot- and cooled-air. Most differences are obviously in the application
software, especially in the way of data analysis and how the derived
crossing points or threshold levels are computed. Each of these
systems employs either one of several general types of fluorescent
probes for detection. There are also big differences how data are
displayed. Some of the limitations of end-point detection in (RT-) PCR
have been assuaged in real-time PCR systems, a number of which are now
on the market. These systems offer many general technical advantages,
including reduced probabilities of variability and contamination, as
well as online monitoring and the lack of need for post reaction
analyses. Further, some of these systems were developed with
contemporary applications such as quantitative PCR, multiplexing, and
high-throughput analysis in mind. Initial template levels can be
calculated by analysing the shape of the curve or by determining when
the signal rises above some threshold value.

A hopefully complete list of the commercially available real-time PCR
systems are overviewed on this page and summarized here =3D>
http://CYCLERS.gene-quantification.info


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GenEx 5 -  A Powerful Tool For qPCR Data Analysis

GenEx is a popular software for qPCR data processing and analysis.
Built in a modular fashion GenEx provides a multitude of
functionalities for the qPCR community, ranging from basic data
editing and management to advanced cutting-edge data analysis. View
our webpage =3D> http://GenEx.gene-quantification.info

Basic data editing and management
Arguably the most important part of qPCR experiments is to pre-process
the raw data into shape for subsequent statistical analyses. The pre-
processing steps need to be performed consistently in correct order
and with confidence. GenEx Standard=92s streamlined and user-friendly
interface ensures mistake-free data handling. Intuitive and powerful
presentation tools allow professional illustrations of even the most
complex experimental designs.

Advanced cutting-edge data analysis
When you need more advanced analyses GenEx Enterprise is the product
for you. Powerful enough to demonstrate feasibility it often proves
sufficient for most users demands. Current features include parametric
and non-parametric statistical tests, Principal Component Analysis,
and Artificial Neural Networks. New features are continuously added to
GenEx with close attention to customers=92 needs.

New features
Sample handling and samples individual biology often contribute to
confounding experimental variability. By using the new nested ANOVA
feature in GenEx version 5 user will be able to evaluate variance
contributions from each step in the experimental procedure. With a
good knowledge of the variance contributions, an appropriate
distribution of experimental replicates can be selected to minimize
confounding variance and maximize the power of the experimental
design! For experiments with complex features, such as for example
multifactorial diseases, analytical relationships and classifications
may not readily be available. The support vector machine feature in
the new version of GenEx is so easy to use that it will make this
advanced supervised classification method easily available to novice
users, while providing access to advanced parameters for experts.

Download a free trail version here =3D> http://GenEx.gene-quantification.in=
fo


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MIQE_qPCR APP for - iOS Universal & Android version

Get help from a special team of experts in qPCR while on the move.
MIQE - qPCR helps you in reviewing scientific works and checking your
own experiments, when qPCR is involved. Check your project's
compliance to MIQE in minutes, have all required references to hand,
and follow qPCR events and news.....
http://www.gene-quantification.de/miqe-qpcr-app-slide-show.pdf

Over 2,500 iOS versions downloaded on http://itunes.apple.com/app/miqe-qpcr=
/id423650002?mt=3D8

MIQE_qPCR Android version - Now available since Nov 2011

https://market.android.com/details?id=3Dcom.biorad.miqeqPCR


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to further scientists and friends who are interested in qPCR !

Best regards,
Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages



If this newsletter is not displayed correctly by your email client,
please use following LINK
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