qPCR NEWS - October 2011 - focus on MIQE and RefGenes -------------------------------------------------------------------------------
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: * MIQE Guidelines are entering "high impact" journals! - http://MIQE-press.gene-quantification.info * RefGenes - a unique tool to find suitable reference genes - http://normalisation.gene-quantification.info * MIQE qPCR APP for iPhone, iPad and iPod - new updated version available - http://MIQE.gene-quantification.info * Join the qPCR 2011 US - http://www.qPCRsymposium.com ----------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following http://qPCRnews.gene-quantification.info ----------------------------------------------------------------------------- MIQE Guidelines are entering "high impact" journals! http://MIQE-press.gene-quantification.info LIFE SCIENCE TECHNOLOGIES - qPCR Innovations and Blueprints Science, 7. October 2011 by Chris Tachibana http://www.sciencemag.org/site/products/lst_20111007.xhtml Quantitative PCR users can rapidly generate large amounts of high- quality data with new instruments and products made possible by microfluidics and miniaturization technology. These platforms are the tools for developing techniques that require extremely high throughput and sensitivity such as digital PCR and single-cell analysis. Researchers are adopting these methods to ask sophisticated questions about genetics and cancer biology as well as to develop novel research and diagnostic assays. As qPCR innovators explore new frontiers and everyday users venture into more complicated workflows, international groups of industry and academic partners are keeping us on the path of best practices. Two consortia (MIQE & SPIDIA) are generating guidelines on the qPCR process - from experimental design and pre- analysis sample collection, to processing data and publishing results. The guidelines are blueprints that ensure reproducibility, validity, and transparency. Routine lab method's accuracy called into question Nature Medicine Vol 16, page 349 (2010) by Catherine Shaffer in Nature Medicine http://www.nature.com/nm/journal/v16/n4/full/nm0410-349.html In 2002, four years after first sparking public controversy over whether the measles, mumps and rubella vaccine causes autism, Andrew Wakefield reported a possible molecular mechanism for the connection. He claimed that a form of irritable bowel disease, which he called autistic enterocolitis, was triggered by the measles virus (Molec. Pathol. 55, 84–90, 2002). That finding, however, was based on a “defective experimental technique,” Stephen Bustin, a molecular biologist at Barts and the London School of Medicine and Dentistry, told a US federal court in 2007. The problem: Wakefield had incorrectly applied the common laboratory protocol known as quantitative real-time polymerase chain reaction (qPCR) to come to his conclusions. Bustin says this faulty lab work is a problem shared by many researchers around the world who have turned to qPCR to measure gene expression. Unlike standard PCR, which can only crudely quantify levels of DNA, the chemistry behind qPCR allows researchers to assess such levels more precisely by comparing sequences of interest against a known reference added to the test tube mix as a control. But the reference genes used in qPCR can vary between experiments and laboratories, which can give misleading results or make it difficult to compare one study to another. As a result of this and other variables in the technique, a majority of scientific papers involving qPCR include flawed methods, say a team of leading qPCR experts. Most qPCR methods, as reported in the literature, are improperly validated and irreproducible, Bustin claims. Last year, he and 11 colleagues published a set of more than 60 individual standards - collectively called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to address this problem ( Clin. Chem. 55, 611–622, 2009 ). “If you look at the literature, you find again and again and again the appalling quality of qPCR protocols,” says Bustin, who this month repeated his call for the scientific community to adopt the MIQE guidelines (Methods 50, 217–226, 2010). “There's no excuse for anyone either not reporting or not doing experiments properly.” The consequence of poor methodology is that many published papers contain erroneous conclusions, says Mikael Kubista, a coauthor of the MIQE guidelines and chief executive of the TATAA Biocenter in Göteborg, Sweden. “The problem is that the technique itself seems so simple and so easy to do, (but) in real life you're analyzing biological samples with complexity.” ----------------------------------------------------------------------------- RefGenes - a unique tool to find suitable reference genes http://www.refgenes.org General problem The choice of suitable reference genes is absolutely crucial in RT- qPCR gene expression analysis. Often, genes from commercial panels don't work well for one's own biological context. Ideally, the expression of reference genes should remain unchanged across samples within the context under study. Solution RefGenes is an online app from Genevestigator that allows users to search for genes that are most stable across a chosen set of samples based on microarray data. This set of samples can be chosen according to experimental conditions or tissue types. For example, if you are performing a RT-qPCR experiment on mouse liver samples, you can use RefGenes to identify the set of genes that are most stable across all microarrays done on mouse liver in Genevestigator. This method offers two major improvements over existing methods because a) it does not narrow down from a small set of genes (e.g. commercial housekeeping gene panels), but looks for novel candidates from a genome-wide set of genes b) it is based on condition-specific stability. The below schema shows how RefGenes can be used in combination with existing approaches to yield valuable reference genes for specific experimental conditions. RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi L, Verbrugge P, Kalaydjieva L, Bleuler S, Laule O, Descombes P, Gruissem W and P Zimmermann BMC Genomics 2011, 12: 156 ----------------------------------------------------------------------------- MIQE_qPCR APP for iPhone, iPad and iPod - iOS Universal new version available! 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 2,000 downloads up to now! http://itunes.apple.com/app/miqe-qpcr/id423650002?mt=8 ----------------------------------------------------------------------------- qPCR 2011 US - http://www.qPCRsymposium.com Mission Center Ballroom, Santa Clara Convention Center, 5001 Great America Parkway, Santa Clara, CA 95054 Symposium Focus * Preanalytics, Standardization and quality control * High throughput expression profiling – digital PCR * Epigenetics, mutation analysis and copy number variation * Molecular diagnostics of complex diseases – detection and profiling of tumor cells * Single-cell and subcellular expression profiling * Non coding RNAs * qPCR in Forensics and AgriBio * Next Generation Sequencing (NGS) * qPCR & NGS experimental design and data mining * Clinical applications of qPCR and NGS Follow the symposium agenda! http://www.qpcrsymposium.com/default.asp?pagecat=C ----------------------------------------------------------------------------- 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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