qPCR NEWS - July 2010 - focus on PCR efficiency

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Thu Jul 22 09:11:58 EST 2010

qPCR NEWS - July 2010 - focus on PCR efficiency

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 - qPCR efficiency estimation
- eConference with high quality 50 talks presented at the
"International qPCR 2010 Symposium in Vienna"
- View our eSeminar and eConference demo videos  =3D>
- qPCR Symposium USA in November 2010
- qPCR Application Workshops  -  and more ... ... ...


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 =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. Different tissues exhibit
different PCR efficiencies, caused by RT inhibitors, PCR inhibitors
and by variations in the total RNA fraction pattern extracted.   read
more ... ... ... Chapter 3 - Quantification strategies in real-time
PCR    pages 87-112   in:  A-Z of quantitative PCR  ( Editor:  S.A.
Bustin )   International University Line (IUL),  La Jolla,  CA,  USA

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


New added publications:

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

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

A new real-time PCR method to overcome significant quantitative
inaccuracy due to slight amplification inhibition.
Guescini M, Sisti D, Rocchi MB, Stocchi L, Stocchi V.;  BMC
Bioinformatics. 2008 30;9: 326

Amplification efficiency: linking baseline and bias in the analysis of
quantitative PCR data.
Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff
MJ, Moorman AF.;  Nucleic Acids Res. 2009 37(6): e45

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 Apr;
50(4): 313-22


qPCR 2010 in Vienna
   International qPCR Symposium & Exhibition
   7-9th April 2010
   Topic:   "The ongoing evolution of qPCR"

BioEPS is now presenting the qPCR 2010 eConference online via video
With our new eConference streaming server a world-wide know-how
transfer becomes possible in a fast and simple way!

During the symposium almost all presented talks (50 in total) were
recorded in excellent sound quality and high movie resolution.
They are available NOW as eConfernce qPCR 2010 on our streaming
server  =3D>  http://eConference.qPCR2010-Vienna.net

You can watch an in-depth demo presentation of our seminars and talks
right here!
=3D> http://eseminars.bioeps.com/player/?action=3Ddemo

Our offers include:

- 20 days free online access to all the talks as often as you want !
- Online access from any computer at any time !
- High resolution streaming technology with great sound quality and
high movie resolution !
- Please take a look at the system requirements for our video streams
before making a purchase.

=3D>  http://eConference.bioeps.com

The qPCR 2010 eConference package contains 50 talks in high resolution
from the qPCR Symposium 2010 in Vienna with the following sessions:

- MIQE and QM strategies in qPCR  (incl. SA Bustin)
- High throughput quantitative PCR =96 digital PCR   (incl. M Kubista, P
- HRM =96 High Resolution Melting - Epigenetics  (incl. C Wittwer, C
- CNA - Circulating nucleic acids    (incl. P Pinzani, J Huggett)
- Single-cell qPCR  (incl. K Livak)
- RNAi - microRNA - siRNA Applications =96 miRNA normalisation (incl. J
Vandesompele, M Castoldi, MW Pfaffl)
- qPCR data analysis - BioStatistics & BioInformatics (incl. J
Hellemans, M Kubista, A Tichopad)

- detailed info about all talks and the speakers =3D> http://sessions.qpcr2=

- To get connected - visit our web shop  =3D>  http://eConference.bioeps.co=


BioEPS GmbH - qPCR Application Workshops

Life Science is still a growing sector and new methods and
technologies are continously developed. Therefore permanent training
and education becomes so important.

With our specific course program we are offering a range of high-
quality course modules, in cooperation with different companies to
give a general and independent overview of existing qPCR technologies
and systems. Our course issues are based on skilled know-how from own
research studies and publications.

Our aim is to point out a critical way of thinking to increase the
quality and outcome of experimental data.

All courses are held regularly in Freising-Weihenstephan, Germany, in
German and English language.
Further customized workshops and specialized trainings will be held as
well across Europe and world-wide.
Workshops are powered by 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://workshops.gene-quantification.info/

Course Occasions 2010:

3-day qPCR Basic Module
2-day BioStatistics & Expression Profiling Module
3-day single-cell qPCR
2-day microRNA qPCR
1-day HRM
2-das qPCR-R data analysis   NEW !
1-day Project Management   NEW !
2-day Quality Management  NEW !

Course dates 2010:

4 - 6  October 2010  (E)   3-day qPCR Basic Module (Mon. - Wed.)
7  October 2010  (E)    1-day HRM Module (Thu.)
11 - 12 October  2010  (E)   2-day qPC-R - data analysis using R
packages (Mon.-Tue.)
18 - 20 October  2010  (E)   3-day single-cell & qPCR (Mon. - Wed.)
21 - 22 October  2010  (E)   2-day Experiment Design  &  qPCR data
processing  (Thu. - Fri.)
8 - 9  November 2010  (E)   2-day microRNA & qPCR (Mon.-Tue.)
29 November - 1 December 2010  (E)   3-day Experiment Design  &  qPCR
data processing  (Mon. - Wed.)
2 - 3 December 2010  (E)   2-day BioStatistics Module (Thu. - Fri.)

Download course brochure 2010 =3D> http://www.gene-quantification.de/bioeps=

Register here =3D> http://site.bioeps.com/index.php?option=3Dcom_seminar&It=

Access to our workshops =3D> http://www.gene-quantification.de/bioeps-acces=


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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