qPCR NEWS - Jan 2011 - Data Analysis & Bioinformatics & qPCR 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 of new publications in qPCR Biostatistics & Bioinformatics - http://bioinformatics.gene-quantification.info - update of new publications in qPCR efficiency calculation - http://efficiency.gene-quantification.info - qPCR 2011 Event - Final Call for TALK and POSTER abstracts - http://CALL.qPCR2011.net ----------------------------------------------------------------------------- Data Analysis and BioInformatics in real-time qPCR How to do successful gene expression analysis using real-time PCR Stefaan Derveaux, Jo Vandesompele, Jan Hellemans; Methods Vol 50, Issue 4, April 2010, in The ongoing Evolution of qPCR edited by Michael W. Pfaffl, Pages 227-230 Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting. Further papers added: - Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula - Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli - Statistical aspects of quantitative real-time PCR experiment design - The Prime Technique - Real-time PCR Data Analysis - Gene expression profiling – Clusters of possibilities - Validation of differential gene expression algorithms: application comparing fold-change estimation to hypothesis testing - Automated validation of polymerase chain reaction amplicon melting curves - Statistical models in assessing fold change of gene expression in real-time RT-PCR experiments - The Importance of Quality Control During qPCR Data Analysis - Error bars in experimental biology - Automatic Genomics: a user-friendly program for the automatic designing and plate loading of medium-throughput qPCR experiments - Interactive analysis of systems biology molecular expression data - Roadmap for developing and validating therapeutically relevant genomic classifiers - Cluster analysis and display of genome-wide expression patterns more info here => http://www.gene-quantification.de/bioinf-subpage3.html -------------------------------------------------------------------------------- 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 more info here => http://efficiency.gene-quantification.info Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar. Regier N & Frey B.; BMC Mol Biol. 2010 11: 57, 8 pages BACKGROUND: RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method. RESULTS: We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method. CONCLUSIONS: As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression. New added publications in 2010 & 2011 => http://efficiency.gene-quantification.info 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 Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8 Schriewer A, Wehlmann A, Wuertz S. J Microbiol Methods. 2011 Jan 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 Enhancement in the efficiency of polymerase chain reaction by TiO2 nanoparticles: crucial role of enhanced thermal conductivity Abdul Khaliq R, Sonawane PJ, Sasi BK, Sahu BS, Pradeep T, Das SK, Mahapatra NR. Nanotechnolog 2010 21(25): 255704 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 2011 Event - 5th international qPCR Symposium & Industrial Exhibition & Application Workshop 28th March - 1st April 2011 in Freising-Weihenstephan, Technical University of Munich, Weihenstephan, Germany qPCR 2011 Event - Final Call for TALK and POSTER abstracts - http://CALL.qPCR2011.net Please submit your abstract here => http://submission.qPCR2011.net On behalf of the Organisation Committee and the Scientific Board it is a great pleasure to invite you to the 5th International qPCR Symposium & Industrial Exhibition & Application Workshop to be held at the Center of Life Science in Freising Weihenstephan, Technische Universität München (Germany). The great international interest in the previous meetings (qPCR 2004, qPCR 2005, qPCR 2005 in Leipzig, qPCR 2007, qPCR 2009, and qPCR 2010 in Vienna) with up to 600 participants coming from 56 countries, and over 40 international companies in the qPCR Industrial Exhibition led us to the decision to repeat the Symposium in spring 2011. => http://www.qPCR2011.net We have set the date for the qPCR 2011 Event to 28th March - 1st April 2011. The event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich is part of the largest Biotech cluster in Europe, located close to the Munich airport in the heart of Bavaria. The focus of the qPCR 2011 Event will be on: Molecular Diagnostics: from single-cells to Next Generation Sequencing Leading academic researchers and industrial contributors in the field will participate in the symposium, which will be an arena for fruitful discussions between researchers of different backgrounds. The Symposium Talks, Poster Sessions, Industrial Exhibition and associated qPCR Application Workshops offer an overview of the present knowledge and future developments in qPCR and gene expression measurement technology and its wide applications. The symposium will focus on 60-70 lectures and more than 100 posters will be presented by internationally recognised experts in their field. The emphasis will be on unbiased, didactic information exchange. Internationally reknown speakers will be participating in a lively and exciting programme enabling the valuable exchange of information in the qPCR field. One third of the talks will be presented by selected invited speakers, one third will be selected from the submitted abstracts and one third will be presented by qPCR related company R&D representatives. All scientific contributions will be published in the qPCR 2011 Symposium Proceedings. Please register here => http://registration.qPCR2011.net Download 1st qPCR 2011 symposium announcement => http://tinyurl.com/CALLqPCR2011 The qPCR 2011 Event contains several parts: * qPCR Symposium March 28th - 30th => talk and poster sessions => confirmed academic and industrial invited speakers * A parallel qPCR Industrial Exhibition March 28th - 30th * Followed by three qPCR application Workshops March 31st - April 1st powered by the TATAA Biocenter & MultiD Sweden - Basic real-time qPCR Application Workshop (2-days) - qPCR data analysis: Biostatistics & Expression Profiling (2-days) - MIQE guidelines (1 day) & Practical primer design (1-day) Symposium Sessions: - Molecular diagnostics in single-cells - High throughput analysis in qPCR & Next Generation Sequencing - MIQE and QM strategies in qPCR - small RNAs qPCR - microRNA and siRNA Applications - Digital PCR & Nano-fluidics - Pre-analytical Steps - qPCR BioStatistics & BioInformatics The scientific organization is managed by international well-known scientists in the field of real-time PCR: - Stephen Bustin, Prof. of Molecular Science QM, School of Medicine, London, UK - Mikael Kubista, Prof. of Biotechnology, TATAA Biocenter, Sweden - Jo Vandesompele, Prof. at the Center of Medical Genetics, University of Ghent, Belgium - Heinrich H.D. Meyer, Prof. of Physiology, Technical University of Munich, Germany - Michael W. Pfaffl, Reader in Physiology, TUM, Weihenstephan, Germany scientific coordinator of the Symposium and the Application Workshops [email protected] Event organization: Dr. Martina Reiter, BioEPS GmbH, Freising, Germany [email protected] We are looking forward to meeting you in March 2011 at the Symposium in Freising-Weihenstephan Michael W. Pfaffl Martina Reiter Symposium Chair BioEPS GmbH Please register here => http://registration.qPCR2011.net Please submit your abstract here => http://submission.qPCR2011.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 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. Copyright © 2005 - 2011 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. 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