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Gene Quantification Newsletter
March  2015

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 www.Gene-Quantification.info
The focus of this newsletter issue is:


Streaming portal

Join the new eConferences streaming webportal -- www.eConferences.de -- Amplify your knowledge!
This portal is dedicated to scientists from the community of qPCR, digital-PCR, NGS and Molecular Diagnostics. You’ll find here all the records from around 200 presentations held at qPCR & NGS Events in the past years. We provide the presentations via movie streaming technology in high quality - high resolution and perfect sound quality in high speed - on any internet browser or mobile device, including Android or iOS.

Optimisation in qPCR

Optimisation &  Standardisation

The reproducibility of biomedical research -- Sleepers awake!
Stephen A. Bustin,
Biomolecular Detection and Quantification, Volume 2, December 2014, Pages 35–42

There is increasing concern about the reliability of biomedical research, with recent articles suggesting that up to 85% of research funding is wasted. This article argues that an important reason for this is the inappropriate use of molecular techniques, particularly in the field of RNA biomarkers, coupled with a tendency to exaggerate the importance of research findings.

To do's and dont's -- The state of RT-quantitative PCR
Firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)

Taylor SC and Mrkusich EM
J Mol Microbiol Biotechnol. 2014 24(1): 46-52

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

... more papers about the MIQE guidelines -- MIQE.Gene-Quantification.info 

Statistics in qPCR

Statistics for Biologists
by www.Nature.com

There is no disputing the importance of statistical analysis in biological research, but too often it is considered only after an experiment is completed, when it may be too late.
This collection highlights important statistical issues that biologists should be aware of and provides practical advice to help them improve the rigor of their work.
Nature Methods' Points of Significance column on statistics explains many key statistical and experimental design concepts. Other resources include an online plotting tool and links to statistics guides from other publishers:  Practical Guides -- Statistics in Biology -- Points of Significance -- other Resources

Methods of integrating data to uncover genotype-phenotype interactions
Ritchie MD, Holzinger ER, Li R, Pendergrass SA, Kim D
Nat Rev Genet. 2015 16(2): 85-97

Recent technological advances have expanded the breadth of available omic data, from whole-genome sequencing data, to extensive transcriptomic, methylomic and metabolomic data. A key goal of analyses of these data is the identification of effective models that predict phenotypic traits and outcomes, elucidating important biomarkers and generating important insights into the genetic underpinnings of the heritability of complex traits. There is still a need for powerful and advanced analysis strategies to fully harness the utility of these comprehensive high-throughput data, identifying true associations and reducing the number of false associations. In this Review, we explore the emerging approaches for data integration - including meta-dimensional and multi-staged analyses - which aim to deepen our understanding of the role of genetics and genomics in complex outcomes. With the use and further development of these approaches, an improved understanding of the relationship between genomic variation and human phenotypes may be revealed.


Next Steps in Reproducibility
PlosOne Blog by Damian Pattinson & Virginia Barbour -- posted: November 13, 2014

In last week’s Nature and Science, the outcome of a meeting convened by NIH, Nature, and Science to discuss the issue of lack of reproducibility in the basic science research literature was published. This meeting, which brought together representatives from publishers (including PLOS), and many representatives from the NIH and other funders, produced a series of principles, Proposed Principles and Guidelines for Reporting Preclinical Research, which were endorsed by a large and diverse group of publishers, associations, and societies including ourselves. The main principles are as follows:

  • Rigorous statistical analysis
  • Transparency in reporting
  • Data and material sharing
  • Consideration of refutations
  • Consider establishing best practice guidelines for image based data and descriptions of biological data.
Optimisation &  Standardisation

Critical Review -- Real-time PCR detection chemistry
Navarro E, Serrano-Heras G, Castaño MJ, Solera J
Clin Chim Acta. 2015 439: 231-250

Checklist for optimization and validation of real-time PCR assays
Raymaekers M, Smets R, Maes B, Cartuyvels R.
J Clin Lab Anal. 2009 23(3): 145-151

Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements
Devonshire AS, Elaswarapu R, Foy CA
BMC Genomics. 2010 24;11: 662

Good Practice Guide for the Application of Quantitative PCR (qPCR)
Nolan T, Huggett J, Sanchez E
Published:  11 October 2013

Chapter 5 - RT-PCR Optimisation Strategies
in PCR Troubleshooting and Optimization - The Essentail Guide edited by Suzanne Kennedy & Nick Oswald
by Martina Reiter & Michael W. Pfaffl
Physiology Weihenstephan, Weihenstephaner Berg 3, 85354 Freising, Germany

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments
David Svec, Ales Tichopad, Vendula Novosadova, Michael W. Pfaffl, Mikael Kubistaa
Biomolecular Detection and Quantification;  available online 11 March 2015

... ... more interesting papers -- Optimisation.Gene-Quantification.info


qPCR, dPCR, NGS -- A journey
Jim F. Huggett, Justin O’Grady, Stephen Bustin
Biomolecular Detection and Quantification, available online 15 January 2015

Scientific conferences fulfill many roles, but one of the most important ones is that they help shape the direction in which a scientific discipline grows by promoting person-to-person exchanges of information, ideas and constructive criticisms between scientists from different backgrounds. This interaction also helps to identify areas of controversy and promotes efforts to address and, it is hoped, resolve them. This year is the 30th anniversary of the publication of the first practical description of the polymerase chain reaction, arguably one of the simplest and the most widely used molecular technology. It also sees the 7th instalment of the Freising PCR meetings www.qPCR-NGS-2015.net, which are the longest established, continuous and most influential conferences in this field and have provided a looking glass for conceptual and technical innovation as well as practical applications of PCR-associated methods.

... ... read more about it -- qPCR.Gene-Quantification.info


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. Download the recent BDQ papers on Elsevier Science Direct.

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 Wittwer, 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 3D Gene 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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