qPCR NEWS May 2009 - focus on RNA integrity ------------------------------------------------------------ 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: - The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments - RNA integrity - latest papers - PowerNest - illuminating error in qPCR experiment design - new TAG CLOUD for better navigation =3D> http://directory.gene-quantifica= tion.info/ - New qPCR workshop modules at the TATAA Biocenter Germany =3D> http://tataa.gene-quantification.info/ - European wide qPCR application workshops =3D> register now ! ---------------------------------------------------------------------------= ----- The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer Clinical Chemistry 2009, 55(4): 611-622 =3D> http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf BACKGROUND: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT: The Minimum Information for Publication of Quantitative Real- Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results. =3D> http://miqe.gene-quantification.info/ ---------------------------------------------------------------------------= ----- As part of the MIQE guidelines - chapter 5.1 - the RNA quality control is an essential step in the quantification process. 5. Nucleic acid quality control 5.1. RNA samples Since it is advisable to use approximately the same amount of RNA for cDNA synthesis when comparing different samples, quantification of RNA in extracted samples is important. However, there are several procedures in common use, including spectrophotometry (Nanodrop), microfluidic analysis (Agilent BioAnalyser, BioRad Experion), capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye detection (Ribogreen); all produce different results making it unwise to compare data obtained using the different methods. The preferred method for RNA quantity determination uses fluorescent RNA binding dyes, for example RiboGreen, which are best for the detection of low target concentrations. In any case, it is advisable to measure all samples using one method only and to report this information. ... ... ... latest papers =3D> http://www.gene-quantification.de/rna-integrity2.html - Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization. - Time course analysis of RNA stability in human placenta. - Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. - A comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration range. - Validation of lab-on-chip capillary electrophoresis systems for total RNA quality and quantity control. - Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. - Optimization of the method of RNA isolation from paraffin blocks to assess gene expression in breast cancer. - Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods. - Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue. - Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. - Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.) - Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer capillary electrophoresis system. - Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. ---------------------------------------------------------------------------= ----- PowerNest - illuminating error in qPCR experiment design PowerNest is a software tool enabling experimenters to explore the effect of sampling on noise propagation throughout qPCR assays. The sampling process is assumed to be comprised of a number of levels; the acquisition of a sample and the preparation of extracted material, reverse-transcription of the mRNA, and the qPCR itself. Given a small set of data, representative of a larger assay, the error at each stage of the experiment is profiled using a nested-ANOVA. Armed with this information, PowerNest allows the experimenter to explore the effects of modifications to the experimental design on the expected total error of the assay. When given the financial cost of replicates at each level, PowerNest will calculate a cost-optimal sampling-plan, delivering an experiment design that will minimise processing error and maximise the statistical resolution of the assay. The software is temporarily undergoing final testing, during which time it has been made available as a free download =3D> http://www.gene-quantification.de/main-bioinf.shtml#powernest PowerNest Poster =3D> http://www.gene-quantification.de/powernest-poster.pd= f ---------------------------------------------------------------------------= ----- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- 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 G=F6teborg, 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: =3D> http://TATAA.gene-quantification.info/ Course Occasions 2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) 3-day single-cell qPCR Module (Mon. - Wed.) 3-day microRNA Module (Mon. - Wed.) 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 13 - 15 Juli 2009 (E) NEW microRNA qPCR (in cooperation with Qiagen) 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 14 - 16 September 2009 (E) NEW single-cell qPCR 19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf =3D> http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pd= f ---------------------------------------------------------------------------= ----- 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 =A9 2005 - 2009 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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