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Gene Quantification Newsletter
September 2014
is sponsored by

     
     

Streamlined and automated NGS workflow


 
  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 domain. The focus of this newsletter issue is:
   


 

 
COMMENT Some cautionary notes on the petite "Holy Grail" of molecular diagnostics
Vandesompele J and Mestdagh P.
Comment in Haematologica. 2014 99(3): 401-402

The “Holy Grail” of molecular diagnostics is the sensitive and specific detection of a disease-associated stable biomarker in non-invasively-acquired patient material. Without realizing it, we may actually have found it. Since the seminal papers by Mitchell et al. (2008) and Chim et al. (2008), many studies have shown that small RNA molecules called microRNAs circulate in the blood in cell-free mode. They appear to be quite abundant in serum and plasma, are stable, and their levels are correlated to disease state, prognosis or response to treatment.
So far, 2578 human mature microRNAs are known (miRBase release 20). Many of these are expressed in a cell type-specific manner, making them ideal candidate biomarkers. MicroRNAs are also useful therapeutic targets; one drug is already in clinical trials and several more are waiting to enter clinical phases. MicroRNAs display exquisite stability in serum or plasma because they are packaged in membrane-encapsulated vesicles or protected by RNA-binding proteins. MicroRNAs also survive in unfavorable physiological conditions, such as repeated freeze-thawing, and long-term storage at room temperature. ... ...
Despite the cautionary notes outlined here, there is a bright future for using circulating RNAs, and in particular microRNAs, because of their tissue specificity and stability, as an embodiment of the long-searched for “Holy Grail” of non-invasive molecular diagnostics.

... more microRNA papers miRNA.gene-quantification.info

 

microRNA QUANTIFICATION ACCURACY & REPEATABILITY

miRQC STUDY

A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability
Redshaw N, Wilkes T, Whale A, Cowen S, Huggett J, Foy CA.
Biotechniques. 2013 54(3): 155-164

MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study
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 S, Ulmanella U, Veeramachaneni V, Staedtler F, Peters T, Guettouche T, Wong L, Vandesompele J.
Nat Methods. 2014 11(8): 809-815

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.

 
microRNA / mRNA
INTERACTION

Walking the interactome to identify human -- miRNA-disease associations through the functional link between miRNA targets and disease genes
Hongbo Shi, Juan Xu, Guangde Zhang, Liangde Xu, Chunquan Li, Li Wang, Zheng Zhao, Wei Jiang, Zheng Guo and Xia Li
BMC Systems Biology 2013, 7:101

Background: MicroRNAs (miRNAs) are important post-transcriptional regulators that have been demonstrated to play an important role in human diseases. Elucidating the associations between miRNAs and diseases at the systematic level will deepen our understanding of the molecular mechanisms of diseases. However, miRNA-disease associations identified by previous computational methods are far from completeness and more effort is needed.
Results: We developed a computational framework to identify miRNA-disease associations by performing random walk analysis, and focused on the functional link between miRNA targets and disease genes in protein-protein interaction (PPI) networks. Furthermore, a bipartite miRNA-disease network was constructed, from which several miRNA-disease co-regulated modules were identified by hierarchical clustering analysis. Our approach achieved satisfactory performance in identifying known cancer-related miRNAs for nine human cancers with an area under the ROC curve (AUC) ranging from 71.3% to 91.3%. By systematically analyzing the global properties of the miRNA-disease network, we found that only a small number of miRNAs regulated genes involved in various diseases, genes associated with neurological diseases were preferentially regulated by miRNAs and some immunological diseases were associated with several specific miRNAs. We also observed that most diseases in the same co-regulated module tended to belong to the same disease category, indicating that these diseases might share similar miRNA regulatory mechanisms.
Conclusions: In this study, we present a computational framework to identify miRNA-disease associations, and further construct a bipartite miRNA-disease network for systematically analyzing the global properties of miRNA regulation of disease genes. Our findings provide a broad perspective on the relationships between miRNAs and diseases and could potentially aid future research efforts concerning miRNA involvement in disease pathogenesis.

Posttranscriptional Regulatory Networks: From Expression Profiling to Integrative Analysis of mRNA and microRNA Data
Swanhild U. Meyer, Katharina Stoecker, Steffen Sass, Fabian J. Theis and Michael W. Pfaffl
Chapter 15  in  Quantitative Real-Time PCR: Methods and Protocols (Methods in Molecular Biology)
by Roberto Biassoni, Alessandro Raso

Protein coding RNAs are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Insights in messenger RNA (mRNA) and microRNA (miRNA) regulatory interactions facilitate the understanding of fi ne-tuning of gene expression and might allow better estimation of protein synthesis. However, in silico predictions of mRNA–microRNA interactions do not take into account the specifi c transcriptomic status of the biological system and are biased by false positives. One possible solution to predict rather reliable mRNA-miRNA relations in the specifi c biological context is to integrate real mRNA and miRNA transcriptomic data as well as in silico target predictions. This chapter addresses the workfl ow and methods one can apply for expression profi ling and the integrative analysis of mRNA and miRNA data, as well as how to analyze and interpret results, and how to build up models of posttranscriptional regulatory networks.

The multilayered complexity of ceRNA crosstalk and competition
Yvonne Tay, John Rinn & Pier Paolo Pandolfi
Nature (2014) 505, 344–352

Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges — they communicate with and co-regulate each other by competing for binding to shared microRNAs, a family of small non-coding RNAs that are important post-transcriptional regulators of gene expression. Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease.

... more papers on integrative analysis integrated-analysis.gene-quantification.info

 

qPCR & NGS 2015
First event announcement

qPCR & NGS 2015 Event
"Advanced Molecular Diagnostics for Biomarker Discovery"
7th international qPCR & NGS Event -- Symposium  &  Industrial Exhibition  &  Application Workshops
23-27 March 2015, in Freising-Weihenstephan, Technical University of Munich, Weihenstephan, Germany
www.qPCR-NGS-2015.net

The great international interest in the previous qPCR & NGS Events from 2004 till 2013 with a constant audience of more than 500 participants from all over the world motivates repeating the success next year in March 2015. We broaden our focus in genomics applications from quantitative RT-PCR, over digital PCR to the latest Next Generation Sequencing technologies. The date for the 7th International qPCR Symposium & Exhibition & Application Workshops is from the 23rd to the 27th March 2015. Parallel to the scientific symposium an industrial exhibition will take place where around 35-40 international companies will be presenting their newest qPCR, dPCR and NGS services and technologies. The symposium will be followed by various qPCR & NGS Workshops taking place March 26th and 27th powered by TATAA Biocenter, Bio-Rad, Genomatix and other leaders in the field.
Event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich are part of the largest Biotech cluster in Europe, located close to the Munich airport (MUC) directly in the heart of Bavaria.

The focus of the qPCR & NGS 2015 Event is "Advanced Molecular Diagnostics for Biomarker Discovery"

The symposium is based on around 60-70 lectures and 100 posters presented by international recognised experts in their application fields. The emphasis will be on unbiased, didactic and scientific information exchange. One third of the talks will be presented by invited speakers, one third of the speakers will be selected from the submitted abstracts and one third will be given to qPCR and NGS company R&D representatives. Various poster sessions will be held in parallel in a separate poster exhibition hall. All scientific contributions will be published in an abstract book (qPCR & NGS 2015 Proceedings incl. ISBN). All talks will be recorded and made public in autumn 2015 via the www.eConferences.de streaming platform together with around 200 talks from qPCR 2010 onwards.

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.

As usual the qPCR Event is structured in three parts: 

  1. Symposium & Poster Sessions, taking place March 23 - 25   Talks.qPCR-NGS-2015.net
  2. Industrial Exhibition with around 40 companies, taking place March 23 - 25   Exhibition.qPCR-NGS-2015.net
  3. qPCR & NGS Application Workshops, taking place March 26 - 27    Workshops.qPCR-NGS-2015.net
 

Symposium Talk & Poster Sessions:

  • Main topic:   Advanced Molecular Diagnostics
  • Main topic:   Biomarker Discovery
  • Main topic:   Next Generation Sequencing (NGS)
     - NGS overview talks - information technology in the era of NGS
     - Pre NGS - sample prep & setup & library generation
     - NGS – new sequencing technologies (e.g. single molecule and pore sequencing)
     - NGS – data analysis (data management, mapping, alignment algorithms, data de novo assembly )
     - NGS – diagnostic applications
  • MicroGenomics & Single-cells diagnostics
  • Circulating Nucleic Acids
  • Molecular Diagnostics in Agriculture, Veterinary Medicine & Environmental Science
  • MIQE & QM & Standardisation strategies in molecular diagnostics
  • Digital PCR  &  Nano-fluidics
  • Non-coding RNAs -- microRNA, small RNAs, long non-coding RNAs
  • qPCR Data Analysis -- BioStatistics & BioInformatics

Register for the symposium and the workshops => Registration.qPCR-NGS-2015.net

Download the latest event flyer => Event-announcement.qPCR-NGS-2015.net

Event location on Google maps  http://goo.gl/maps/3p3gx

View our previous talks and the event trailer on our streaming server -- www.eConferences.de

 
 

NEW Journal -- "Biomolecular Detection and Quantification" -- Call for submissions

Biomolecular Detection and Quantification (BDQ) is an open access, peer-reviewed international journal dedicated to championing excellence in molecular study design, measurement, data analysis and reporting. Its focus is on the application of qualitative and quantitative molecular methodologies to all areas of clinical and life sciences. The journal has two main aims:

  • to provide a forum for discussion and recommendation of guidelines designed to improve the accuracy of molecular measurement, its data analysis and the transparency of its subsequent reporting;
  • to publish molecular biology based studies that adhere to best practice guidelines, both current and future.

Benefits of publishing open access with Elsevier's Biomolecular Detection and Quantification - www.journals.elsevier.com/biomolecular-detection-and-quantification/

  • Peer-reviewed journal indexed by Scopus and supporting ORCID and Crossref to help maintain your publication record.
  • A renowned and respected editorial team with extensive knowledge of your research field. BDQ was established by a group of scientists based on their experience developing and publishing the Minimum Information for Publication of Quantitative Real-Time (and Digital) PCR Experiments (the MIQE and digital MIQE guidelines).
  • Reaching key audiences with 10 million active users per month using our publishing platform ScienceDirect.
  • Choice of user licenses so authors can decide how best to publish open access.
  • A discount on the publication fee for a limited time.

For more information about publishing Open Access with Elsevier, including funding body arrangements, institutional agreements and more, visit: www.elsevier.com/openaccess

We look forward to receiving your paper!

Kind regards,

The Editors
Stephen Bustin, Jim Huggett, Justin O'Grady, Michael W. Pfaffl, Carl Witwer, Ron Cook

View full editorial board

 

GenEx 6:
real-time PCR data analysis software
 

GenEx 6 available - The most powerful tool for complex qPCR data analysis
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. Download GenEx User Guide

TATAA Biocenter, Europe´s leading provider of genomic services using quantitative real-time PCR (qPCR), and MultiD Analyses, Europe’s prime software developer for the analysis of multivariate data, release GenEx version 6 for accurate analysis of qPCR data compliant with current Clinical Laboratory Standards Institute (CLSI) guidelines.

The new analyses in GenEx 6 include: Estimating PCR efficiency, testing for outliers, testing linear model, estimating dynamic range, estimating random error, estimating limit of detection, estimating limit of quantification, estimating concentrations of unknowns, evaluation of precision, and verification of precision. For all estimates confidence intervals are calculated.

Additional new features include: Transcript distribution for single cell analysis, Survival Analysis, Receiver Operator Characteristics (ROC), Wizard for ProSeek protein analysis, and reader for 3DGene microRNA analysis.

View our GenEx webpage and download a FREE GenEx 6 trial version => GenEx.Gene-Quantification.info

Join all GenEx talks by Prof. Mikael Kubista held at recent qPCR & NGS Events =>  www.eConferences.de/multid/

 

Best regards,

Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages

 

   

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