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 ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ ---------------------------------------------------------------------------= ----- 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 =E2=80=98real expression ratio=E2=80=99 compared to NO ef= ficiency correction. Small efficiency differences between target and reference gene generate false expression ratio, and the researcher over- or under-estimates the =E2=80=98real=E2=80=99 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. ---------------------------------------------------------------------------= ----- Several methods are described in the literature to calculate real-time PCR efficiency: http://efficiency.gene-quantification.info/ Real-Time PCR: A Review of Approaches to Data Analysis (Rebrikov & Trofimov 2006) Effect of DNA damage on PCR ampli=EF=AC=81cation efficiency with the relati= ve 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) ---------------------------------------------------------------------------= ----- 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 http://infoportal.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, ...etc.. Please submit your qPCR event here =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- 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 =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= ..pdf ---------------------------------------------------------------------------= ----- 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 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 ---------------------------------------------------------------------------= ----- qPCR WORKSHOP TATAA Biocenter Germany - qPCR Application workshops http://tataa.gene-quantification.info/ 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=C3=B6teborg, 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=C3=B6teborg 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=C3=B6teborg 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=C3=B6teborg 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 =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 http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl and powered by BioScience Events. Copyright =C2=A9 2005 - 2008 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=3DSUBSCRIBE