qPCR Newsletter July 2008 - main focus on qPCR optimisation

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Mon Jul 28 09:48:57 EST 2008

qPCR Newsletter July 2008 - main focus on qPCR optimisation

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 - Directory page
- Steps and variables of a successful mRNA quantification using real-
time RT-PCR
- How to optimize your qPCR
- qPCR Event calendar 2008:  meetings  &  application workshops

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Steps and variables of a successful mRNA quantification using real-
time RT-PCR



How to optimize your quantitative real-time PCR PROTOCOL - Current
problems in quantitative real-time RT-PCR.
by T. Nolan, RE. Hands & SA Bustin;  Nature Protocols (2006) Vol. 1,
No. 3; p1559-1582

The real-time reverse transcription polymerase chain reaction (RT-
qPCR) addresses the evident requirement for quantitative data analysis
in molecular medicine, biotechnology, microbiology and diagnostics and
has become the method of choice for the quantification of mRNA.
Although it is often described as a =91=91gold=92=92 standard, it is far fr=
being a standard assay. The significant problems caused by variability
of RNA templates, assay designs and protocols, as well as
inappropriate data normalization and inconsistent data analysis, are
widely known but also widely disregarded. The widespread use of this
technology has resulted in the development of numerous protocols that
generate quantitative data using: fresh, frozen or archival FFPE
(formalin-fixed, paraffinembedded) samples,
whole-tissue biopsies, microdissected samples, single cells, tissue
culture cells,
total or mRNA,
- a range of different cDNA priming strategies,
- different enzymes or enzyme combinations,
- assays of variable efficiency, sensitivity and robustness,
- diverse detection chemistries, reaction conditions, thermal cyclers
- individual analysis and reporting methods.
This obvious lack of standardization at every step of the assay
(Figure 1) is exacerbated by significant differences in sample
processing, use of controls, normalization methods and quality control
management and has serious implications for the reliability, relevance
and reproducibility of RT-qPCR. An overview of the considerations
relating to procedures and alternative steps for carrying out the RT-
qPCR reaction is shown in Figure 2.


New papers qPCR optimisation

- Evaluation of probe chemistries and platforms to improve the
detection limit of real-time PCR.
- Diagnostic PCR: validation and sample preparation are two sides of
the same coin.
- Faster quantitative real-time PCR protocols may lose sensitivity and
show increased variability.
- Real-time RT-PCR: considerations for efficient and sensitive assay
- How to reduce primer dimers (3 papers)
- Standardization and quality control of PCR analyses.
- Standardization of real-time PCR gene expression data from
independent biological replicates.
- Control of Contamination Associated with PCR and Other Amplification
- Enhancing the efficiency of a PCR using gold nanoparticles.
- Increasing Detection of Polymerase Chain Reaction (PCR) by Isolation
of PCR Products (IPCRp).
- Increased efficiency of genetic profiling through quantity and
quality assessment of fluorescently   labeled oligonucleotide primers.
- Primers with 5' flaps improve real-time PCR.
- A real-time polymerase chain reaction-based evaluation of cDNA
synthesis priming methods.
- Comparison of quantitative competitive polymerase chain reaction=96
enzyme-linked immunosorbent assay with LightCycler-based polymerase
chain reaction for measuring cytomegalovirus DNA in patients after
hematopoietic stem cell transplantation.
- Performance evaluation of thermal cyclers for PCR in a rapid cycling
- Sensitivity comparison of real-time PCR probe designs on a model DNA
- Real-time RT-PCR and SYBR Green I melting curve analysis for the
identification of Plum pox virus strains C, EA, and W: Effect of
amplicon size, melt rate, and dye translocation.
- Comparison of two standardisation methods in real-time quantitative
RT-PCR to follow Staphylococcus aureus genes expression during in
vitro growth.


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-



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 =3D>  http://www.gene-quantification.de/meetings.html#benelux

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 =3D>  http://www.gene-quantification.de/qpcr_benelux_poster=


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

More info =3D>  http://www.gene-quantification.de/meetings.html#qpcr_usa

- 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
- 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
- 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



TATAA Biocenter Germany - qPCR Application workshops

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 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 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:  http://tataa.gene-quantification.info/

Course Occasions summer and autumn 2008:

25-29 Aug  Prague    qPCR Core Module + Practical Biostatistics
8-12 Sep  G=F6teborg   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 G=F6teborg   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 G=F6teborg    qPCR Core Module + Biostatistics
15-19 Dec Prague    RNA Isolation + Expression Profiling and Data
Download list of TATAA courses in autumn and fall 2008

Please register here  =3D>  http://www.tataa.com/Courses/Courses.html


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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