qPCR NEWS December 2008

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Wed Dec 17 04:03:08 EST 2008

qPCR NEWS December 2008


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:

- Final Call for TALKS & POSTERS for qPCR 2009 Event   =3D> http://www.qPCR=
- European wide qPCR application workshops =3D>  register now !
- REST 2008 update
- PCR efficiency page updated with the newest papers in the field
- Online translation service of the Gene Quantification
=3D> http://translation.gene-quantification.info/


Dear qPCR NEWS-Reader,

Best Wishes for the Holiday Season

and a

Happy and Successful New Year 2009 !


REST 2008 update REST 2008 builds on its predecessor REST 2005 with
significant improvements to randomization algorithms. This new
revision introduces alternative data inputs such as single run
efficiency and amplification take-off point, alleviating the need to
set amplification plot thresholds.

REST 2008 is a standalone software package for analyzing gene
expression using real-time amplification data. The software addresses
issues surrounding the measurement of uncertainty in expression ratios
by introducing randomization and bootstrapping techniques. New
confidence intervals for expression levels also allow measurement of
not only the statistical significance of deviations but also their
likely magnitude, even in the presence of outliers. Whisker box plots
provide a visual representation of variation for each gene,
highlighting potential issues such as distribution skew. REST 2008
builds on its predecessor REST 2005 with significant improvements to
randomization algorithms. This new revision introduces alternative
data inputs such as single sample efficiency and amplification take-
off point, alleviating the need to set amplification plot thresholds.


What's NEW since REST 2005 ?

NEW - REST-RG mode
A new method of input has been introduced, allowing users to copy and
paste results from the Rotor-Gene software's Comparative Quantitation
analysis. This is an alternative to importing standard curve and CT
results. See REST-RG Mode chapter for more details.

NEW - Whisker-Box Plots Exportable
Whisker-Box plots can now be exported by right-clicking on the graph.

NEW - Improved randomisation
Improvements to the randomisation algorithms have been made, making
confidence intervals much tighter, and p-values more accurate. In
previous versions the pair-wise fixed reallocation was incorrectly
matching the gene of interest CT with the incorrect reference CT, this
issue has been rectified in REST 2008.

NEW - Handling of standard curve variation
REST 2008 no longer takes into account the variation of the standard
curve and implements improvements to the calculation of confidence
intervals and p-values. In previous versions, the software would
randomly pick two points from the standard curve and calculate an
efficiency based on that. However there is a situation when two points
are chosen that lie close to each other on the standard curve, this
can cause a bogus efficiency which adds unnecessary outliers to the
random distribution. We now calculate the efficiency by determining
the line of best fit for the standard curve, this efficiency is used
through the randomisation process.

Download =3D> REST 2008 software

Download =3D>  Manual  REST 2008 version 2.0.7

Slide Show REST-2008

The new stand-alone software versions REST 2008 was programmed and
designed by Matthew Herrmann, David Chiew, and Birgit Speller working
at Corbett Research (Sydney, Australia) and Michael W. Pfaffl,
Technical University of Munich, Germany.


update in determination of real-time PCR amplification efficiency

taken from Chapter 3:   Quantification strategies in real-time PCR
in:  A-Z of quantitative PCR  ( Editor:  S.A. Bustin ) International
University Line (IUL), La Jolla, CA, USA

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 =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.
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

Find lots of useful papers here =3D> http://efficiency.gene-quantification.=

latest papers published in 2008:

- A kinetic-based sigmoidal model for the polymerase chain reaction
and its application to high-capacity absolute quantitative real-time
PCR (Rutledge & Stewart, 2008)
- A new real-time PCR method to overcome significant quantitative
inaccuracy due to slight amplification inhibition (Guescini et al.,
- 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)
- Critical evaluation of methods used to determine amplification
efficiency refutes the exponential character of real-time PCR
(Rutledge & Stewart 2008)
- A new method for robust quantitative and qualitative analysis of
real-time PCR (Shain & Clemens, 2008)
- CAmpER - Real-time PCR analysis software - Calculation of
Amplification Efficiencies for RT-PCR experiments is a tool for the
automatic analysis of real time PCR experiments. Automatic analysis,
annotation and storage of real-time PCR experiments performed with
different qPCR systems, currently the LightCycler 2, LC480, RotorGene
and the Opticon.


online translation

Since October 2008 we provide an online translation service of the
Gene Quantification pages to several languages. Please recognize this
is an automatic and robotic based translation service, and therefore
we can give NO guarantee about the generated content. It should help
to understand the "rough" content of the Gene Quantification pages,
but still the original is the ENGLISH version:




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.
=3D>  Papers / Protocols / Methods / Databases / Alets / Feeds / Books /
Forums / E-mail / Directory



Upcoming Events World-wide academic and commercial qPCR Events

Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars,
qPCR Education Program, etc.
Please submit your qPCR event here  =3D>  events from gene-


qPCR 2009 EVENT - 9 - 13 March 2009

more news here  =3D>  http://www.qPCR2009.net
download event FLYER  =3D>  http://www.bioeps.com/qpcr2009/qPCR-2009-3rd-an=

It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at
the qPCR 2009 event in an own session =9325th Anniversary of PCR=94

List of confirmed speakers  =3D>  http://speakers.qpcr2009.net/
An industrial exhibition with the 30 world leading companies will be
held during the qPCR Symposium March 9-11  =3D>  http://exhibition.qpcr2009=
Our sponsors  =3D>  http://sponsors.qpcr2009.net/

Please register until 31st December to get the early registartion fee
=3D>  http://registration.qpcr2009.net/

Keynote lectures

The Pioneer in PCR   Kary B. Mullis   1993 Nobel Prize in Chemistry
- 25th Anniversary of PCR

Stephen A. Bustin
Professor of Molecular Science, Institute of Cell and Molecular
Science, Queen Mary's School of Medicine and Dentistry, University of
London, UK

- A new qPCR assay for the detection of Clostridium difficile
- MIQE- guidelines for publication of qPCR data

Ken Livak
Senior Scientific Fellow, Fluidigm Corporation,San Francisco, CA, US
- Moving from qPCR Assays to qPCR Arrays.



BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops

At the TATAA Biocenter Germany we offer qPCR application workshops, a
3-day qPCR Core Module and a 2-day qPCR Biostatistics Module.  All
courses are held regularly in G=F6teborg, 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 workshops are held in cooperation with BioEPS
GmbH, located at the campus of the Technical University of Munich, in
Freising-Weihenstephan, very close to the Munich Airport (MUC). For
more information and registration, please see our web page:
=3D> http://TATAA.gene-quantification.info/

Course Occasions 2008/2009:
3-day qPCR Core Module  (Mon. - Wed.)
2-day BioStatistics Module (Thu. - Fri.)
single-cell qPCR Module  (Mon. - Wed.)

26 - 30th January 2009 (E)    in Freising, Germany, English language
26 - 30th January 2009 (E)    in Freising, Germany, English language
16 - 18 February 2009 (E)     in Freising, NEW microRNA & qPCR
12 - 13 March 2009 (E)        various 2-day courses at the qPCR 2009
                              =3D>  http://workshops.qpcr2009.net/


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


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