qPCR Newsletter July 2008 - main focus on qPCR optimisation

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Michael Pfaffl
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qPCR Newsletter July 2008 - main focus on qPCR optimisation

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 ‘‘gold’’ standard, it is far from 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 and
- 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 design.
- 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 Reactions.
- 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–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 condition.
- Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid.
- 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.
=> 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 => eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%65%76%65%6e%74%73%40%67%65%6e%65%2d%71%75%61%6e%74%69%66%69%63%61%74%69%6f%6e%2e%69%6e%66%6f%22%3e%65%76%65%6e%74%73%40%67%65%6e%65%2d%71%75%61%6e%74%69%66%69%63%61%74%69%6f%6e%2e%69%6e%66%6f%3c%2f%61%3e%27%29%3b'))



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


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

More info => 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 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



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

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 => 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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