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Gene Quantification Newsletter
October 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:


digital PCR

Digital PCR (dPCR)

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.

new and hot
dPCR papers

Digital PCR as a novel technology and its potential implications for molecular diagnostics.
Huggett JF and Whale A.
Clin Chem. 2013 59(12): 1691-1693
Comment on -- Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury.

Analysis of extracellular RNA by digital PCR.
Takahashi K, Yan IK, Kim C, Kim J, Patel T
Front Oncol. 2014 4: 129 -- eCollection 2014
The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Quantification of cell-free DNA in normal and complicated pregnancies -- overcoming biological and technical issues.
Manokhina I, Singh TK, Peñaherrera MS, Robinson WP.
PLoS One. 2014 9(7): e101500 -- eCollection 2014

Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.
Liao GJ, Gronowski AM, Zhao Z.
Clin Chim Acta. 2014 428: 44-50

methodological aspects of dPCR

Direct elicitation of template concentration from quantification cycle (Cq) distributions in digital PCR.
Mojtahedi M, Fouquier d'Hérouël A, Huang S.
Nucleic Acids Res. 2014 42(16): e126

A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples.
Sedlak RH, Kuypers J, Jerome KR.
Diagn Microbiol Infect Dis. 2014 S0732-8893 (14) 00366-6

Impact of variance components on reliability of absolute quantification using digital PCR.
Jacobs BK, Goetghebeur E, Clement L.
BMC Bioinformatics. 2014 Aug 22;15: 283

dPCR applications Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.
Yang R, Paparini A, Monis P, Ryan U.
Int J Parasitol. 2014 S0020-7519 (14) 00226-4

Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer.
Li N, Ma J, Guarnera MA, Fang H, Cai L, Jiang F.
J Cancer Res Clin Oncol. 2014 Jan;140(1): 145-150

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.
Fu GK, Wilhelmy J, Stern D, Fan HC, Fodor SP.
Anal Chem. 2014 Mar 18;86(6): 2867-2870

GMO quantification: valuable experience and insights for the future.
Milavec M, Dobnik D, Yang L, Zhang D, Gruden K, Zel J.
Anal Bioanal Chem. 2014 Oct;406(26): 6485-6497


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

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    Exhibition is almost sold out !
  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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