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qPCR newsletter August 2008 with focus on PCR efficiencydetermination

qPCR newsletter August 2008 with focus on PCR efficiencydetermination - Protocols and Methods Forum

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Old 08-14-2008, 01:38 PM
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Default qPCR newsletter August 2008 with focus on PCR efficiencydetermination



qPCR newsletter August 2008 with focus on PCR efficiency determination





Dear researcher,
dear Gene Quantification page reader,

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

- Update - PCR efficiency page
- qPCR Event calendar 2008
- qPCR application workshops

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If this newsletter is not displayed correctly by your email client,
please use following link:
[Only registered users see links. ]
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Determination of real-time PCR amplification efficiency

Individual samples generate different and individual fluorescence
histories in kinetic RT-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 a major impact on the fluorescence
history and the accuracy of the calculated expression result and is
critically influenced by PCR reaction components. Efficiency
evaluation is an essential marker in gene quantification procedure.
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 housekeeping 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 ‘real expression ratio’ 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 ‘real’ initial mRNA amount.
The assessment of the exact amplification efficiencies of target and
reference genes must be carried out before any calculation of the
normalized gene expression is done. LightCycler Relative Expression
Software, Q-Gene, REST and REST-XL software applications allow the
evaluation of amplification efficiency plots. Different tissues
exhibit different PCR efficiencies, caused by RT inhibitors, PCR
inhibitors and by variations in the total RNA fraction pattern
extracted.

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Several methods are described in the literature to calculate real-time
PCR efficiency:

[Only registered users see links. ]

Real-Time PCR: A Review of Approaches to Data Analysis (Rebrikov &
Trofimov 2006)
Effect of DNA damage on PCR amplification efficiency with the relative
threshold cycle method (Sikorsky et al., 2004)
A kinetic model of quantitative real-time polymerase chain reaction
(Mehra S, Hu WS., 2005)
A kinetic-based sigmoidal model for the polymerase chain reaction and
its application to high-capacity absolute quantitative real-time PCR
(Rutledge & Stewart, 2008)
Model based analysis of real-time PCR data from DNA binding dye
protocols (Alvarez et al., 2007)
A new real-time PCR method to overcome significant quantitative
inaccuracy due to slight amplification inhibition (Guescini et al.,
2008)
Highly accurate sigmoidal fitting of real-time PCR data by introducing
a parameter for asymmetry (Spiess et al., 2008)
qpcR: an R package for sigmoidal model selection in quantitative real-
time polymerase chain reaction analysis (Ritz & Spiess, 2008)
Comparing Algorithms for Calculating Amplification Efficiencies of
Real-Time PCR (Arikawa et al, 2007)
Absolute and relative real-time PCR in the quantification of tst gene
expression among methicillin-resistant Staphylococcus aureus:
evaluation by two mathematical models (Chini et al, 2007)
Enhancing the efficiency of a PCR using gold nanoparticles (Li et al,
2006)
PCR inhibition by reverse transcriptase leads to an overestimation of
amplification efficiency (Suslov 2005)
Relative quantification of mRNA: comparison of methods currently used
for real-time PCR data analysis (Cikos et al, 2007)
Technical Note - Evaluation of Real-Time PCR Amplification
Efficiencies to Detect PCR Inhibitors (Kontanis & Reed, 2006)


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With the new qPCR INFO PORTAL and all the presented tools we will help
you with to find the right information about qPCR and related topics
in Molecular Biology in the literature and in the World Wide Web.
=> Papers / Protocols / Methods / Databases / Alets / Feeds / Books /
Forums / E-mail / Directory

[Only registered users see links. ]


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Upcoming Events World-wide academic and commercial qPCR Events
[Only registered users see links. ]

Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars,
qPCR Education Program, ...etc..
Please submit your qPCR event here => events@gene-
quantification.info


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qPCR SYMPOSIUM BENELUX

The prominent and still growing place taken by real-time quantitative
PCR in applied and fundamental research and clinical diagnostics
almost appears obvious. However, it is clear that contributions made
by various scientists and companies in the field during the last
decade rendered this technology useful and affordable for many users.

More info => [Only registered users see links. ]

Importantly, the qPCR domain is still in constant evolution, making it
sometimes hard to stay informed about new methodological approaches or
original studies using the real-time PCR. Therefore, we have scheduled
a one day "Benelux qPCR Symposium" on October 6th 2008, giving the
opportunity to the scientific community to get informed and discuss
various aspects of real-time PCR (including but not limited to new
applications, assay optimization and validation, new technologies,
etc.). Scientific talks, posters sessions and industrial booths will
be at the menu.

Download poster => [Only registered users see links. ]


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qPCR Symposium USA
10. - 13. November 2008
Clarion Hotel San Francisco Airport, Millbrae, CA , USA

More info => [Only registered users see links. ]

- High throughput platforms: High throughput applications, real-time
RT-PCR arrays, digital PCR
- Forthcoming technologies: Immuno PCR, Methylation sensitive PCR,
SNP analysis, High resolution melt, microRNA detection, - Multiplex
technologies
- Single-cell qPCR: Pre-amplification techniques, sub-cellular PCR,
Expression heterogeneity, laser microdissection, FACS sorting,
Enrichment of rare cells
- Multimarker diagnostics: Disease markers, Tissue specific markers,
Cancer markers, Stem cells, Differentiation markers, Cancer stem cells
- Real-time PCR Expression Profiling: multivariate and multiway
expression profiling, temporal expression profiling, spatiotemporal
maps
- Pre-analytical Steps: Sampling technologies, Extraction methods,
Reverse Transcription, Quality Control, Standards, Standard Operating
Procedures, Interlaboratory Exercises
- Normalization & Standardization: Normalization strategies,
Reference genes, Spikes, Standard curves, multiplexing, inter-run
calibrators, quantification strategies, mRNA degradation
- Data management and data treatment: software applications, data
mining, data visualization, biostatistics, multivariate statistics



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qPCR WORKSHOP

TATAA Biocenter Germany - qPCR Application workshops

[Only registered users see links. ]

At the TATAA Biocenter Germany we offer qPCR application workshops,
the 3-day Core Module and a 2-day Biostatistics Module. qPCR courses
are held in regularly in Göteborg, Sweden, in English and in Freising-
Weihenstephan, Germany, in German and English, and in Prague, Czech
Republic in English and Czech.
Depending on the occasion the workshop language and the different
prices may apply. Further customized workshops and specialized
trainings will be held as well across Europe and world-wide. TATAA
Biocenter Germany courses are held in cooperation with the Institute
of Physiology, located at the Technical University of Munich, in
Freising-Weihenstephan, near Munich, very close to the Munich Airport
(MUC). For more information and to register for the qPCR application
workshops, please see our web page: [Only registered users see links. ]

Course Occasions summer and autumn 2008:

25-29 Aug Prague qPCR Core Module + Practical Biostatistics
8-12 Sep Göteborg Sample Preparation + qPCR Core Module
15 - 19 Sep Freising Germany qPCR Core Module + Biostatisticsy
(English language )
13-17 Oct Prague RNA Isolation + qPCR Core Module + HRM
13-17 Oct Freising Germany qPCR Core Module + Biostatistics (Kurs
wird in DEUTSCH gehalten, German language)
27-31 Oct Göteborg qPCR Core Module + HRM + Biostatistics
17-21 Nov Prague qPCR Core Module + Practical Biostatistics
24-28 Nov Freising Germany qPCR Core Module + Biostatistics
(English language)
1-5 Dec Göteborg qPCR Core Module + Biostatistics
15-19 Dec Prague RNA Isolation + Expression Profiling and Data
Analysis
Download list of TATAA courses in autumn and fall 2008

Please register here => [Only registered users see links. ]

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Forward Please send the qPCR NEWS to further scientists and friends
who are interested in qPCR !


Best regards,

Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages
[Only registered users see links. ]


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