Question: qPCR NEWS - December 2008
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: - Final Call for TALKS & POSTERS for qPCR 2009 Event => - European wide qPCR application workshops => register now ! - REST 2008 update - PCR efficiency page updated with the newest papers in the field - Online translation service of the Gene Quantification => ---------------------------------------------------------------------- ---------- Dear qPCR NEWS-Reader, Best Wishes for the Holiday Season and a Happy and Successful New Year 2009 ! ---------------------------------------------------------------------- ---------- REST 2008 update REST 2008 builds on its predecessor REST 2005 with significant improvements to randomization algorithms. This new revision introduces alternative data inputs such as single run efficiency and amplification take-off point, alleviating the need to set amplification plot thresholds. REST 2008 is a standalone software package for analyzing gene expression using real-time amplification data. The software addresses issues surrounding the measurement of uncertainty in expression ratios by introducing randomization and bootstrapping techniques. New confidence intervals for expression levels also allow measurement of not only the statistical significance of deviations but also their likely magnitude, even in the presence of outliers. Whisker box plots provide a visual representation of variation for each gene, highlighting potential issues such as distribution skew. REST 2008 builds on its predecessor REST 2005 with significant improvements to randomization algorithms. This new revision introduces alternative data inputs such as single sample efficiency and amplification take- off point, alleviating the need to set amplification plot thresholds. What's NEW since REST 2005 ? NEW - REST-RG mode A new method of input has been introduced, allowing users to copy and paste results from the Rotor-Gene software's Comparative Quantitation analysis. This is an alternative to importing standard curve and CT results. See REST-RG Mode chapter for more details. NEW - Whisker-Box Plots Exportable Whisker-Box plots can now be exported by right-clicking on the graph. NEW - Improved randomisation Improvements to the randomisation algorithms have been made, making confidence intervals much tighter, and p-values more accurate. In previous versions the pair-wise fixed reallocation was incorrectly matching the gene of interest CT with the incorrect reference CT, this issue has been rectified in REST 2008. NEW - Handling of standard curve variation REST 2008 no longer takes into account the variation of the standard curve and implements improvements to the calculation of confidence intervals and p-values. In previous versions, the software would randomly pick two points from the standard curve and calculate an efficiency based on that. However there is a situation when two points are chosen that lie close to each other on the standard curve, this can cause a bogus efficiency which adds unnecessary outliers to the random distribution. We now calculate the efficiency by determining the line of best fit for the standard curve, this efficiency is used through the randomisation process. Download => REST 2008 software Download => Manual REST 2008 version 2.0.7 Slide Show REST-2008 The new stand-alone software versions REST 2008 was programmed and designed by Matthew Herrmann, David Chiew, and Birgit Speller working at Corbett Research (Sydney, Australia) and Michael W. Pfaffl, Technical University of Munich, Germany. ---------------------------------------------------------------------- ---------- update in determination of real-time PCR amplification efficiency taken from Chapter 3: Quantification strategies in real-time PCR in: A-Z of quantitative PCR ( Editor: S.A. Bustin ) International University Line (IUL), La Jolla, CA, USA 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. 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. Find lots of useful papers here => http://efficiency.gene- latest papers published in 2008: - A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR (Rutledge & Stewart, 2008) - 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) - Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR (Rutledge & Stewart 2008) - A new method for robust quantitative and qualitative analysis of real-time PCR (Shain & Clemens, 2008) - CAmpER - Real-time PCR analysis software - Calculation of Amplification Efficiencies for RT-PCR experiments is a tool for the automatic analysis of real time PCR experiments. Automatic analysis, annotation and storage of real-time PCR experiments performed with different qPCR systems, currently the LightCycler 2, LC480, RotorGene and the Opticon. ---------------------------------------------------------------------- ---------- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: ---------------------------------------------------------------------- ---------- 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 => events@gene- ---------------------------------------------------------------------- ---------- qPCR 2009 EVENT - 9 - 13 March 2009 more news here => download event FLYER => -3rd-announcement.pdf It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the qPCR 2009 event in an own session “25th Anniversary of PCR” List of confirmed speakers => An industrial exhibition with the 30 world leading companies will be held during the qPCR Symposium March 9-11 => Our sponsors => Please register until 31st December to get the early registartion fee => Keynote lectures The Pioneer in PCR Kary B. Mullis 1993 Nobel Prize in Chemistry - 25th Anniversary of PCR Stephen A. Bustin Professor of Molecular Science, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London, UK - A new qPCR assay for the detection of Clostridium difficile - MIQE- guidelines for publication of qPCR data Ken Livak Senior Scientific Fellow, Fluidigm Corporation,San Francisco, CA, US - Moving from qPCR Assays to qPCR Arrays. ---------------------------------------------------------------------- ---------- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held 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 workshops are held in cooperation with 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: => Course Occasions 2008/2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) single-cell qPCR Module (Mon. - Wed.) 26 - 30th January 2009 (E) in Freising, Germany, English language 26 - 30th January 2009 (E) in Freising, Germany, English language 16 - 18 February 2009 (E) in Freising, NEW microRNA & qPCR 12 - 13 March 2009 (E) various 2-day courses at the qPCR 2009 Event => ---------------------------------------------------------------------- ---------- 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 ---------------------------------------------------------------------- ---------- If this newsletter is not displayed correctly by your email client, please use following link: 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 - 2008 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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