qPCR NEWS - December 2011 - focus on qPCR efficiency ------------------------------------------------------------------------
Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: * qPCR efficiency calculation - sub-domain updated - http://Efficiency.gene-quantification.info * real-time PCR Cycler - sub-domain updated - http://CYCLERS.gene-quantification.info * GenEx version 5 - download a free trial version - http://GenEx.gene-quantification.info * MIQE qPCR APP for IOS and Android is available - http://MIQE.gene-quantification.info 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 real-time 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 'the 'major impact' on the fluorescence history and the accuracy of the calculated expression result and is critically influenced by PCR reaction components. The efficiency evaluation is an essential marker in gene quantification procedure and one of the important characteristicum of the MIQE guidelines. 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 reference 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 ‘real expression ratio’ 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 ‘real’ initial mRNA amount. Find the latest literature about real-time PCR efficiency determination: Efficiency of the Polymerase Chain Reaction. Booth CS, Pienaar E, Termaat JR, Whitney SE, Louw TM, Viljoen HJ. Chem Eng Sci. 2010 65(17): 4996-5006. pcrEfficiency: a Web tool for PCR amplification efficiency prediction. Mallona I, Weiss J, Marcos EC. BMC Bioinformatics. 2011 12: 404 Experimental Validation of a Fundamental Model for PCR Efficiency. Louw TM, Booth CS, Pienaar E, Termaat JR, Whitney SE, Viljoen HJ. Chem Eng Sci. 2011 Apr 15;66(8): 1783-1789. Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR. Lievens A, Van Aelst S, Van den Bulcke M, Goetghebeur E. Nucleic Acids Res. 2011 Nov 18. Validation of kinetics similarity in qPCR. Bar T, Kubista M, Tichopad A. Nucleic Acids Res. 2011 Oct 19. A mechanistic model of PCR for accurate quantification of quantitative PCR data. Boggy GJ, Woolf PJ. Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America. PLoS One. 2010 5(8): e12355. 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 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 Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR. Rutledge RG, Stewart D. PLoS One. 2010 5(3): e9731. 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 50(4): 313-22 ... ... and more => http://Efficiency.gene-quantification.info ----------------------------------------------------------------------------- UPDATE - real-time PCR Cycler On http://CYCLERS.gene-quantification.info the most prominent real- time PCR cycler are described. In the cycler descriptions the specifications and the advantages of the displayed systems are shown. Which real-time platform meets your requirements best, depends on your research application. Some of the systems are designed for research with low capacities and others are for high-throughput applications, most in combination with pipetting robots. Most of them use solid-block for thermal cycling, other use hot- and cooled-air. Most differences are obviously in the application software, especially in the way of data analysis and how the derived crossing points or threshold levels are computed. Each of these systems employs either one of several general types of fluorescent probes for detection. There are also big differences how data are displayed. Some of the limitations of end-point detection in (RT-) PCR have been assuaged in real-time PCR systems, a number of which are now on the market. These systems offer many general technical advantages, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for post reaction analyses. Further, some of these systems were developed with contemporary applications such as quantitative PCR, multiplexing, and high-throughput analysis in mind. Initial template levels can be calculated by analysing the shape of the curve or by determining when the signal rises above some threshold value. A hopefully complete list of the commercially available real-time PCR systems are overviewed on this page and summarized here => http://CYCLERS.gene-quantification.info ----------------------------------------------------------------------------- GenEx 5 - A Powerful Tool For qPCR Data Analysis GenEx is a popular software for qPCR data processing and analysis. Built in a modular fashion GenEx provides a multitude of functionalities for the qPCR community, ranging from basic data editing and management to advanced cutting-edge data analysis. View our webpage => http://GenEx.gene-quantification.info Basic data editing and management Arguably the most important part of qPCR experiments is to pre-process the raw data into shape for subsequent statistical analyses. The pre- processing steps need to be performed consistently in correct order and with confidence. GenEx Standard’s streamlined and user-friendly interface ensures mistake-free data handling. Intuitive and powerful presentation tools allow professional illustrations of even the most complex experimental designs. Advanced cutting-edge data analysis When you need more advanced analyses GenEx Enterprise is the product for you. Powerful enough to demonstrate feasibility it often proves sufficient for most users demands. Current features include parametric and non-parametric statistical tests, Principal Component Analysis, and Artificial Neural Networks. New features are continuously added to GenEx with close attention to customers’ needs. New features Sample handling and samples individual biology often contribute to confounding experimental variability. By using the new nested ANOVA feature in GenEx version 5 user will be able to evaluate variance contributions from each step in the experimental procedure. With a good knowledge of the variance contributions, an appropriate distribution of experimental replicates can be selected to minimize confounding variance and maximize the power of the experimental design! For experiments with complex features, such as for example multifactorial diseases, analytical relationships and classifications may not readily be available. The support vector machine feature in the new version of GenEx is so easy to use that it will make this advanced supervised classification method easily available to novice users, while providing access to advanced parameters for experts. Download a free trail version here => http://GenEx.gene-quantification.info ----------------------------------------------------------------------------- MIQE_qPCR APP for - iOS Universal & Android version Get help from a special team of experts in qPCR while on the move. MIQE - qPCR helps you in reviewing scientific works and checking your own experiments, when qPCR is involved. Check your project's compliance to MIQE in minutes, have all required references to hand, and follow qPCR events and news..... http://www.gene-quantification.de/miqe-qpcr-app-slide-show.pdf Over 2,500 iOS versions downloaded on http://itunes.apple.com/app/miqe-qpcr/id423650002?mt=8 MIQE_qPCR Android version - Now available since Nov 2011 https://market.android.com/details?id=com.biorad.miqeqPCR ----------------------------------------------------------------------------- Please forward this qPCR NEWS http://api.addthis.com/oexchange/0.8/forward/email/offer?url=http://qPCRnews.gene-quantification.info&title=Join+our+monthly+newsletter+on&username=qPCR-NEWS&email_template=&lng=en-us to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages 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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