qPCR NEWS - August 2012 - focus on single-cell qPCR ---------------------------------------------------------------------
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: * UPDATE of various new papers using single-cell PCR technologies in qPCR and NGS - http://singlecell.Gene-Quantification.info * Second announcement qPCR & NGS Symposium in March 2013 - http://www.qPCR-NGS-2013.net * GenEx - a powerful tool for qPCR data analysis - download a free trial version - http://GenEx.gene-quantification.info ----------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following http://qPCRnews.gene-quantification.info ----------------------------------------------------------------------------- UPDATE of various new papers using single-cell PCR technologies in qPCR and NGS => http://singlecell.Gene-Quantification.info Single-cell molecular-biology is a relatively new scientific branch in biology. The first single-cell analysis were involved in the characterization of mitochondrial DNA in 1988. Single-cell DNA analysis, in particular genomic DNA, is important and may be informative in the analysis of genetics of cell clonality, genetic anticipation and single-cell DNA polymorphisms. Nowadays for most sceientists the quantitative transcriptomics in a single-cell is much more important, and the analytical method of choice is the quantitative real-time RT-PCR. In single-cell biology the absolute abundance of particular mRNAs or microRNAs and their up- or down-regulation in a single cell, compared to their neighbour cells, is the goal. The need for quantitative single-cell mRNA analysis is evident given the vast cellular heterogeneity of all tissue cells and the inability of conventional RNA methods, like northern blotting, RNAse protection assay or classical block RT-PCR, to distinguish individual cellular contributions to mRNA abundance differences. new papers Method of the year - Methods to watch - Special feature Single-cell methods - Improved single-cell methods are helping to unravel biological complexity We present important methods and areas of methodological development worth watching in the coming years. Nature Methods - VOL.9 NO.1 - JANUARY 2012 - 35 The heterogeneity of cells in culture and in organisms poses a challenge for many experi-mental measurements. Population measure-ments are necessarily averages, masking the behavior of minority subpopulations and effectively blinding researchers to possibly interesting differences between cells.The alternative is to make measure-ments on single cells. Methodologically speaking, this, too, is challenging on sever-al fronts. Molecular analyses, whether on a particular macromolecule or at an ‘omic’ scale, can be difficult (or even impossible) to accomplish on the amount of mate-rial extracted from one cell. Methods with increased sensitivity are therefore in demand. Throughput is also a bottle-neck. Basing firm conclusions on single-cell measurements means that one must be able to quickly and accurately analyze many cells. Finally, it is often necessary to analyze single cells in a mul-tiplexed fash-ion, either because the cells exist in a heteroge-neous pop-ulation or because one wants to measure many parameters at the same time.There continue to be methodologi-cal advances on all of these fronts. Mass cytometry, for instance - in which iso-topes are used as antibody labels instead of fluorescent probes—considerably extends the multiplexing capabilities of flow cytometry (Science 332, 687–695; 2011). Is the measurement of gene expression, digital reverse-transcriptase PCR in a microfluidics device makes it possible to simultaneously monitor the expres-sion of hundreds of genes in hundreds of single cells. As demonstrated in a recent study of tumor heterogeneity, this can be combined with single cell sorting and with statistical clustering methods to begin to dissect the cellular subpopulations that constitute a tissue (Nat. Biotechnol. 29, 1120–1127; 2011). -------------------------------------------------------------------------------- Cell Biology. Using cell-to-cell variability - a new era in molecular biology Pelkmans L. Science. 2012 Apr 27;336(6080): 425-426 -------------------------------------------------------------------------------- Genomic analysis at the single-cell level Kalisky T, Blainey P, Quake SR. Annu Rev Genet. 2011;45: 431-445 Studying complex biological systems such as a developing embryo, a tumor, or a microbial ecosystem often involves understanding the behavior and heterogeneity of the individual cells that constitute the system and their interactions. In this review, we discuss a variety of approaches to single-cell genomic analysis. -------------------------------------------------------------------------------- A single molecule view of gene expression Larson DR, Singer RH, Zenklusen D. Trends Cell Biol. 2009 (11): 630-637 Analyzing the expression of single genes in single cells appears minimalistic in comparison to gene expression studies based on more global approaches. However, stimulated by advances in imaging technologies, single-cell studies have become an essential tool in understanding the rules that govern gene expression. This quantitative view of single-cell gene expression is based on counting mRNAs in single cells, monitoring transcription in real time, and visualizing single proteins. Parallel advances in mathematical models based on stochastic, discrete descriptions of biochemical processes have provided crucial insights into the underlying cellular mechanisms that control expression. The view that has emerged is rooted in a probabilistic understanding of cellular processes that quantitatively explains both the mean and the variation observed in gene-expression patterns among single cells. Thus, the close coupling between imaging and mathematical theory has established single-cell analysis as an essential branch of systems biology. -------------------------------------------------------------------------------- Single-cell gene-expression profiling and its potential diagnostic applications Stahlberg A, Kubista M, Aman P. Expert Rev Mol Diagn. 2011 (7): 735-740 Gene-expression profiling has been successfully applied in various diagnostic applications, but its full capacity is yet to be realized. Samples are generally prepared from a mixture of different cells that are present in unknown proportions. Cells are, in many aspects, unique in their characteristics and this heterogeneity confounds the expression profile. The development of new and robust techniques to measure gene expression in single cells opens new avenues in molecular medicine. Today, gene-expression profiles of individual cells can be measured with high precision and accuracy, identifying different cell types as well as revealing heterogeneity among cells of the same kind. Here, we review practical aspects of single-cell gene-expression profiling using reverse transcription quantitative real-time PCR and its potential use in diagnostics. ----------------------------------------------------------------------------- New papers using single-cell PCR technologies in qPCR and NGS - http://singlecell.Gene-Quantification.info - Multimarker gene analysis of circulating tumor cells in pancreatic cancer patients: a feasibility study - Mammalian genes are transcribed with widely different bursting kinetics - Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy - Quantification noise in single cell experiments - Identifying single-cell molecular programs by stochastic profiling - High-throughput microfluidic single-cell RT-qPCR - RNA-Seq analysis to capture the transcriptome landscape of a single cell - Development and applications of single-cell transcriptome analysis - Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples - mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients - Comprehensive qPCR profiling of gene expression in single neuronal cells - Single cell transcriptomics of neighboring hyphae of Aspergillus niger - RT-qPCR based quantitative analysis of gene expression in single bacterial cells - Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges - Molecular characterization of circulating tumor cells in breast cancer: challenges and promises for individualized cancer treatment - Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR - Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines - High throughput single cell expression profiling: Taking a closerlook on biological response - Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry - Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes ----------------------------------------------------------------------------- Second announcement qPCR & NGS Symposium in Freising-Weihenstephan 18-22 March 2013 http://www.qPCR-NGS-2013.net Download the latest Event Flyer => http://tinyurl.com/qPCR-NGS-2013 On behalf of the Organisation Committee and the Scientific Board it is a great pleasure to invite you to the 6th International qPCR & NGS 2013 Event. The event is divided in a 3-day scientific Symposium with an Industrial Exhibition and various 2-day Application Workshop to be held at the Center of Life Science in Freising Weihenstephan, Technische Universität München (Germany). The great international interest in the previous meetings ( qPCR 2004 to qPCR 2011 ) led us to the decision to repeat the Symposium in spring 2013. We expect 500-600 participants coming from all over the world, in 2011 we could welcome participants from 56 contries, and roughly 30-40 international companies in the qPCR Industrial Exhibition. We have set the date for the qPCR & NGS 2013 Event to 18th - 22nd March 2013. The event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in Freising Weihenstephan, Germany (Google Maps link or Google Earth link). The TUM and the Biotech region around Munich is part of the largest Biotech cluster in Europe, located close to the Munich airport in the heart of Bavaria. The focus of the qPCR & NGS 2013 Event will be on: Next Generation Thinking in Molecular Diagnostics Leading academic researchers and industrial contributors in the field will participate in the symposium, which will be an arena for fruitful discussions between researchers of different backgrounds. The Symposium Talks, Poster Sessions, Industrial Exhibition and associated qPCR & NGS Application Workshops offer an overview of the present knowledge and future developments in qPCR, next generation sequencing and gene expression measurement technology and its wide applications in research. The symposium will focus on 70 lectures and a huge poster exhibition will be presented by internationally recognised experts in their field. The emphasis will be on unbiased, didactic information exchange. Internationally renown speakers will be participating in a lively and exciting programme enabling the valuable exchange of information in the qPCR and Next Generation Sequencing field. One third of the talks will be presented by selected invited speakers, one third will be selected from the submitted abstracts and one third will be presented by qPCR & NGS related company R&D representatives. All scientific contributions will be published in the Symposium Proceedings (ISBN to be announced). Full papers from selected invited academic and industrial speakers and application notes from industrial speakers will be published in a METHODS special issue “Transcriptional Biomarkers” edited by Michael W. Pfaffl (published January 2013). At the meeting all participants will get a free hard cover of this special Methods issue. Please have a look to our previous issue => “The ongoing evolution of qPCR” METHODS special qPCR Vol 50 issue 4 (April 2010) Please register using the Internet based ConfTool registration and submission platform => http://registration.qPCR-NGS-2013.net ----------------------------------------------------------------------------- Symposium Talk and Poster sessions: http://sessions.qpcr-ngs-2013.net/ - Main Topic: Molecular diagnostics - Main Topic: Next Generation Sequencing (NGS) - Main Topic: Transcriptional Biomarkers - High throughput analysis in qPCR - Systems biology - Single-cells diagnostics - MIQE & QM strategies in qPCR - non-coding RNAs - microRNA, siRNA, long non-coding RNAs - Digital PCR & Nano-fluidics - Pre-analytical Steps - BioStatistics & BioInformatics - qPCR & NGS data analysis - Lunch Seminars: - qBASEplus - data analysis lunch seminar - GenEx - data analysis lunch seminar - NGS data analysis lunch seminars - more to be announced........ View our qPCR 2011 event trailer on YouTube => http://www.youtube.com/watch?v=cp8WwPyLW8Y Download the latest Event Flyer => http://tinyurl.com/qPCR-NGS-2013 Please register using the Internet based ConfTool registration and submission platform => http://registration.qPCR-NGS-2013.net ----------------------------------------------------------------------------- GenEx 5 - A Powerful Tool For qPCR Data Analysis Download a free trail version here => http://GenEx.gene-quantification.info 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 GenEx PDF user guides: * GenEx user guide * GenEx user guide - Exiqon Wizard * GenEx user guide - Roche Wizard ----------------------------------------------------------------------------- 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. 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