qPCR NEWS - July 2010 - focus on PCR 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 - qPCR efficiency estimation - eConference with high quality 50 talks presented at the "International qPCR 2010 Symposium in Vienna" - View our eSeminar and eConference demo videos => http://eseminars.bioeps.com/player/?action=demo - qPCR Symposium USA in November 2010 - qPCR Application Workshops - and more ... ... ... -------------------------------------------------------------------------------- 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 ‘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. 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. Different tissues exhibit different PCR efficiencies, caused by RT inhibitors, PCR inhibitors and by variations in the total RNA fraction pattern extracted. read more ... ... ... Chapter 3 - Quantification strategies in real-time PCR pages 87-112 in: A-Z of quantitative PCR ( Editor: S.A. Bustin ) International University Line (IUL), La Jolla, CA, USA Find lots of useful papers here => http://efficiency.gene-quantification.info -------------------------------------------------------------------------------- New added publications: 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 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 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 2010 in Vienna International qPCR Symposium & Exhibition 7-9th April 2010 http://www.qpcr2010-vienna.net/ Topic: "The ongoing evolution of qPCR" BioEPS is now presenting the qPCR 2010 eConference online via video stream! With our new eConference streaming server a world-wide know-how transfer becomes possible in a fast and simple way! During the symposium almost all presented talks (50 in total) were recorded in excellent sound quality and high movie resolution. They are available NOW as eConfernce qPCR 2010 on our streaming server => http://eConference.qPCR2010-Vienna.net You can watch an in-depth demo presentation of our seminars and talks right here! => http://eseminars.bioeps.com/player/?action=demo Our offers include: - 20 days free online access to all the talks as often as you want ! - Online access from any computer at any time ! - High resolution streaming technology with great sound quality and high movie resolution ! - Please take a look at the system requirements for our video streams before making a purchase. => http://eConference.bioeps.com The qPCR 2010 eConference package contains 50 talks in high resolution from the qPCR Symposium 2010 in Vienna with the following sessions: - MIQE and QM strategies in qPCR (incl. SA Bustin) - High throughput quantitative PCR – digital PCR (incl. M Kubista, P Day) - HRM – High Resolution Melting - Epigenetics (incl. C Wittwer, C Orlando) - CNA - Circulating nucleic acids (incl. P Pinzani, J Huggett) - Single-cell qPCR (incl. K Livak) - RNAi - microRNA - siRNA Applications – miRNA normalisation (incl. J Vandesompele, M Castoldi, MW Pfaffl) - qPCR data analysis - BioStatistics & BioInformatics (incl. J Hellemans, M Kubista, A Tichopad) - detailed info about all talks and the speakers => http://sessions.qpcr2010-vienna.net - To get connected - visit our web shop => http://eConference.bioeps.com -------------------------------------------------------------------------------- BioEPS GmbH - qPCR Application Workshops Life Science is still a growing sector and new methods and technologies are continously developed. Therefore permanent training and education becomes so important. With our specific course program we are offering a range of high- quality course modules, in cooperation with different companies to give a general and independent overview of existing qPCR technologies and systems. Our course issues are based on skilled know-how from own research studies and publications. Our aim is to point out a critical way of thinking to increase the quality and outcome of experimental data. All courses are held regularly in Freising-Weihenstephan, Germany, in German and English language. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. Workshops are powered by BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page => http://workshops.gene-quantification.info/ Course Occasions 2010: 3-day qPCR Basic Module 2-day BioStatistics & Expression Profiling Module 3-day single-cell qPCR 2-day microRNA qPCR 1-day HRM 2-das qPCR-R data analysis NEW ! 1-day Project Management NEW ! 2-day Quality Management NEW ! Course dates 2010: 4 - 6 October 2010 (E) 3-day qPCR Basic Module (Mon. - Wed.) 7 October 2010 (E) 1-day HRM Module (Thu.) 11 - 12 October 2010 (E) 2-day qPC-R - data analysis using R packages (Mon.-Tue.) 18 - 20 October 2010 (E) 3-day single-cell & qPCR (Mon. - Wed.) 21 - 22 October 2010 (E) 2-day Experiment Design & qPCR data processing (Thu. - Fri.) 8 - 9 November 2010 (E) 2-day microRNA & qPCR (Mon.-Tue.) 29 November - 1 December 2010 (E) 3-day Experiment Design & qPCR data processing (Mon. - Wed.) 2 - 3 December 2010 (E) 2-day BioStatistics Module (Thu. - Fri.) Download course brochure 2010 => http://www.gene-quantification.de/bioeps-course-programm-2010.pdf Register here => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6 Access to our workshops => http://www.gene-quantification.de/bioeps-access.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. 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