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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 domain. The focus of this newsletter issue is:
Hot papers in the field of digital PCR - http://digital-PCR.gene-quantification.info
First event announcement -- qPCR & NGS 2015 Event -- "Advanced Molecular Diagnostics for Biomarker Discovery" - www.qPCR-NGS-2015.net
If this newsletter is not displayed correctly by your email client, please follow qPCRnews.gene-quantification.info ----------------------------------------------------------------------------- Digital PCR (dPCR) digital-PCR.gene-quantification.info dPCR is a refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA, or RNA). The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants, point mutations, and it is routinely used for clonal amplification of samples for "next-generation sequencing." The first digital-PCR paper: Quantitation of targets for PCR by use of limiting dilution Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA. Biotechniques. 1992 13(3): 444-449 We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample. ----------------------------------------------------------------------------- More hot and new digital PCR papers => digital-PCR.gene-quantification.info
How to Make More Published Research True John P. A. Ioannidis Plos Medicine - Published: October 21, 2014 DOI: 10.1371/journal.pmed.1001747 Summary Points:
Currently, many published research findings are false or exaggerated, and an estimated 85% of research resources are wasted.
To make more published research true, practices that have improved credibility and efficiency in specific fields may be transplanted to others which would benefit from them—possibilities include the adoption of large-scale collaborative research; replication culture; registration; sharing; reproducibility practices; better statistical methods; standardization of definitions and analyses; more appropriate (usually more stringent) statistical thresholds; and improvement in study design standards, peer review, reporting and dissemination of research, and training of the scientific workforce.
Selection of interventions to improve research practices requires rigorous examination and experimental testing whenever feasible.
Optimal interventions need to understand and harness the motives of various stakeholders who operate in scientific research and who differ on the extent to which they are interested in promoting publishable, fundable, translatable, or profitable results.
Modifications need to be made in the reward system for science, affecting the exchange rates for currencies (e.g., publications and grants) and purchased academic goods (e.g., promotion and other academic or administrative power) and introducing currencies that are better aligned with translatable and reproducible research.
Mestdagh P, Hartmann N, Baeriswyl L, Andreasen D, Bernard N, Chen C, Cheo D, D'Andrade P, DeMayo M, Dennis L, Derveaux S, Feng Y, Fulmer-Smentek S, Gerstmayer B, Gouffon J, Grimley C, Lader E, Lee KY, Luo S, Mouritzen P, Narayanan A, Patel S, Peiffer S, Rüberg S, Schroth G, Schuster D, Shaffer JM, Shelton EJ, Silveria 9, Ulmanella U, Veeramachaneni V, Staedtler F, Peters T, Guettouche T, Vandesompele J
Nature Methods 11, 809–815 (2014)
MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
PCR and qPCR Webinar Series -- A six webinar series on PCR techniques This six webinar series on PCR will be based on MIQE guidelines. MIQE, Minimum Information for Publication of Quantitative-Real Time PCR, helps scientists to design and report reliable and reproducible qPCR results. They help others to understand and replicate already conducted experiments. In this webinar series we will discuss step by step critical points which need to be taken into consideration in order to acquire reliable data. Topics will include: - Basic principles of PCR, qPCR and ddPCR - qPCR Assay Design - Importance of Sample Quality in qPCR Analysis - Improving Assay Quality by Optimization and Validation - Data Analysis - Troubleshooting - See more at: http://www.sigmaaldrich.com/life-science/learning-center/customer-education/pcr-webinar-series.html
Marina received her BSc at Tartu University in Estonia at the Department of Cell and Molecular Biology. In 2001 she moved to Sweden and started her MSc in Molecular Genetics at Department of Plant Biology in Swedish University of Agricultural Sciences. Marina completed her PhD at Department of Genetics and Pathology in Rudbeck Laboratory at Uppsala University. Her thesis was dealing with epigenetic changes of transcription factor genes in haematopoietic tumours. For the last 5 years working as an application specialist in the technical and application support team, providing customers with: assay designs and evaluations, seminars and workshops.
Anders Bergkvist, PhD Application Specialist, Sigma Custom products
Dr. Bergkvist has a strong multidisciplinary academic and industry background with a M.Sc on Engineering Physics from Chalmers University of Technology and a Ph.D. in Biochemistry from Goteborg University in Goteborg and a postdoc in Bioinformatics from Harvard Medical School in Boston. The common theme of his work has been applications of computational tools to biologically relevant tasks. At present he is providing customers with design support and developing new business and marketing opportunities as an Application Specialist at Sigma Life Science.
Tania Nolan, PhD The Gene Team-Consultant for Sigma-Aldrich
Tania Nolan is founder and CEO of The Gene Team Ltd; an international consortium of expert Life Scientists who provide educational and project support to all researchers. She has an international reputation for expertise in the field of mRNA quantification using RT-qPCR and much more importantly, for being able to troubleshooting absolutely any problem! She edited PCR Technologies: Current Innovations with Stephen Bustin and was a significant co-author of the specialist textbook “The A - Z of Quantitative PCR” (ed S.A. Bustin; IUL Press). Tania is an enthusiastic lecturer and has presented several plenary lectures and chaired sessions at major international meetings. Teaching is a particular passion for Tania and she regularly organizes qPCR workshops worldwide. She has an active publication record and regularly contributes to the scientific literature, mainly addressing aspects of quality control of qPCR and qRT-PCR experiments. Tania gained a first class honors degree and was awarded the Excellence in Research Prize from undergraduate studies (University of Salford) and then a PhD in genetics from Manchester University, UK. She took up an AstraZeneca Research Fellowship to study the genetic regulation in breast cancer before moving to Stratagene to support the launch of their qPCR program. Tania lead the Sigma Aldrich Custom Products technical and application support team and was awarded an Honorary Senior Lecturer position at Manchester University in 2014. Outside of science, Tania is a keen clarinettist and plays in a local orchestra as well as small ensemble groups. While on lecture tours, she travels with her clarinets and practices whenever possible.