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Gene Quantification Newsletter
December 2016

is sponsored by

     
     

Streamlined and automated NGS workflow 

   
  Dear researcher,
dear Gene Quantification page reader,

Our newsletter informs about the latest news in gene expression profiling using qPCR and related methods, which are compiled and summarised on www.Gene-Quantification.info
The focus of this qPCR NEWS issue is:
   


 

Streaming portal


Join the updated
eConferences streaming webportal
www.eConferences.de -- Amplify your knowledge in  qPCR, dPCR and NGS!
This streaming portal is dedicated to scientists from the community of qPCR, digital PCR, Next Generation Sequencing (NGS), MicroGenomics (MG) and Molecular Diagnostics (MDx). You’ll find here all the records of 280 presentations held at qPCR & NGS and MG Events in the past years – qPCR 2010 in Vienna  to qPCR & NGS 2015 in Freising-Weihenstephan.
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.

 
BDQ
Special Issue
on digital-PCR

Biomolecular Detection and Quantification, Volume 10, Pages 1-50 (Dec 2016)
Special Issue on digital-PCR

edited by Valerie Taly and Jim Huggett
digital-PCR.Gene-Quantification.info

Editorial - Special issue on dPCR
Valerie Taly, Jim Huggett
Biomolecular Detection and Quantification, Volume 10, Page 1

Digital PCR, a technique for the future
We are very pleased to be able to bring you this special issue of Biomolecular Detection and Quantification which focuses on digital PCR (dPCR). The underpinning method of dPCR, which was coined in 1999 (1), actually predates qPCR (2) and is a powerful technique that could offer improved sensitivity, precision and reproducibility (3). Now is an exciting time for this method and there are numerous examples of where the improved reproducibility can be applied clinically and where the superior sensitivity and precision could enable measurements to be performed that are simply not possible using PCR or, in many cases, sequencing.
In this special issue we have selected seven manuscripts that discuss and present dPCR in a variety of subjects. Dhillon et al. present a new application of dPCR in the form of a proximity ligation assay (PLA) opening the possibility of using limiting dilution to improve the detection and quantification of proteins which, in this case focussed on Clostridium difficile toxins, are important markers for disease. Matthew Butchbach presents a review on the application of dPCR as a robust method to identify genes associated with paediatric-onset disorders. This review also highlights how dPCR can offer a powerful technology to track changes in genomic biomarkers with disease progression. He also argues that dPCR has the potential to become the tool of choice for the verification of mutations identified by next generation sequencing, copy number determination and also for non-invasive prenatal screening.
The next four manuscripts deal with the application and analysis dPCR. Whale et al. discuss multiplexing by dPCR and describe the different approaches that can be applied highlighting the unique approaches on offer by dPCR. They also report and name a characteristic of dPCR, namely partition specific competition (PSC), that must be considered when applying thresholds to multiplex assays that use the same primers but different probes, as is common when measuring single nucleotide variants or polymorphisms. Debski and Garstecki describe how to design dPCR experiments to ensure desired precision is achieved when dealing with patient samples. This is an important and frequently neglected consideration when discussing the performance of any molecular method. Jones and colleagues present a short report that investigates the dynamic range of dPCR, which is often reported as being at a disadvantage when compared with qPCR. In this study they demonstrate that if you have enough partitions it is possible to perform dPCR with a dynamic range of up to six orders of magnitude, which is approaching that of qPCR. Finally the manuscript by Madic et al. describes application of the first three colour dPCR instrument for multiplex analysis of three mutations of the EGFR gene. This droplet-based platform applies a unique sample partition format by employing a “2D droplet array” that can be directly imaged following the PCR reaction.
The last article deals with accuracy, reliability and reproducibility in the context of nucleic acid quantification and highlights how dPCR could be used to quantify reference materials. Bhat and Emslie also discuss how the use of reference materials and certified reference materials, already established when measuring many biochemical analytes, could support the traceable analysis of molecular targets such as BCR-ABL1 (4). While dPCR is already being used to quantify such materials, they highlight the fact that further work is required to better understand sources of bias and uncertainty.
We hope you enjoy these articles which illustrate the potential of this fairly new and unique molecular method. We are of the opinion that dPCR offers considerable potential as a method that will advance clinical research and routine diagnosis and could become the method of choice in areas such as precision and personalised healthcare. This special issue will add to the increasing body of literature reporting on the use of digital PCR in every day laboratory practises and offer solutions to some of remaining challenges and pitfalls that could be encountered.

References:
(1)   B. Vogelstein, K.W. Kinzler; Digital PCR; Proc. Natl. Acad. Sci. U. S. A., 96 (16) (1999), pp. 9236–9241
(2)   A.A. Morley; Digital PCR: a brief history; BDQ, 1 (1) (2014), pp. 1–3
(3)   J.F. Huggett, S. Cowen, C.A. Foy; Considerations for digital PCR as an accurate molecular diagnostic tool Clin. Chem., 61 (1) (2015), pp. 79–88
(4)   H. White, et al.; A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR; Leukemia, 29 (2) (2015), pp. 369–376

 
 

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B
Harvinder S. Dhillon, Gemma Johnson, Mark Shannon, Christina Greenwood, Doug Roberts, Stephen Bustin
Biomolecular Detection and Quantification, Volume 10, Pages 2-8

Applicability of digital PCR to the investigation of pediatric-onset genetic disorders
Matthew E.R. Butchbach
Biomolecular Detection and Quantification, Volume 10, Pages 9-14

Fundamentals of multiplexing with digital PCR
Pages 15-23
Alexandra S. Whale, Jim F. Huggett, Svilen Tzonev
Biomolecular Detection and Quantification, Volume 10, Pages 15-23

Designing and interpretation of digital assays: Concentration of target in the sample and in the source of sample
Pawel R. Debski, Piotr Garstecki
Biomolecular Detection and Quantification, Volume 10, Pages 24-30

Digital PCR dynamic range is approaching that of real-time quantitative PCR
Gerwyn M. Jones, Eloise Busby, Jeremy A. Garson, Paul R. Grant, Eleni Nastouli, Alison S. Devonshire, Alexandra S. Whale
Biomolecular Detection and Quantification, Volume 10, Pages 31-33

Three-color crystal digital PCR
J. Madic, A. Zocevic, V. Senlis, E. Fradet, B. Andre, S. Muller, R. Dangla, M.E. Droniou
Biomolecular Detection and Quantification, Volume 10, Pages 34-46

Digital polymerase chain reaction for characterisation of DNA reference materials
Somanath Bhat, Kerry R. Emslie
Biomolecular Detection and Quantification, Volume 10, Pages 47-49

... and more interesting digital-PCR Papers and review => digital-PCR.Gene-Quantification.info

 
Call for Abstracts

CALL for TALK and POSTER Abstracts @ qPCR dPCR & NGS 2017 => CALL.qPCR-dPCR-NGS-2017.net

 

Join
8th Gene Quantification Event
in April 2017

qPCR dPCR & NGS 2017

qPCR dPCR & NGS 2017
8th international Gene Quantification Event
Symposium & Industrial Exhibition & Application Workshops

Main topics:   Liquid Biopsy, Integrative Big Data Analysis, Biomarker Signature … and beyond
3 - 7 April 2017, in Freising-Weihenstephan,
School of Life Sciences, Technical University of Munich, Weihenstephan, Germany
www.qPCR-dPCR-NGS-2017.net

Keynote Speakers & Scientific Event Announcement => Announcement.qPCR-dPCR-NGS-2017.net

Confirmed Speakers & talk Topics => Talks.qPCR-dPCR-NGS-2017.net 

Event Promotion Trailer => www.eConferences.de/qPCR-dPCR-NGS-2017/

Scientific Symposium Sessions:

  • Liquid Biopsy & Circulating Nucleic Acids
  • Advanced Molecular Diagnostics
  • Integrative Big Data Analysis
  • Biomarker Signatures
  • Digital PCR
  • Non-coding RNAs -- microRNA, isomiRs, small RNAs, long non-coding RNAs
  • MicroGenomics & Single-Cells Diagnostics
  • Next Generation Sequencing (NGS)
  • Molecular Diagnostics in Life Science
  • MIQE & QM & Standardisation Strategies in Molecular Diagnostics

Register & Submit your scientific contribution (talk or poster abstracts) => Registration.qPCR-dPCR-NGS-2017.net

 

GenEx 6.1

The most powerful tool for complex qPCR data analysis

Compliant with MIQE and CLSI guidelines

GenEx offers advanced methods to analyze real-time qPCR data with simple mouse clicks

Download a FREE GenEx 6.1 trial version => 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. GenEx 6.1 – the software compliant with MIQE and CLSI guidelines

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, Hierarchical Cluster Analysis (HCA), Heatmap, Principal Component Analysis (PCA, 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 GenEx 6.1 trail version => GenEx.gene-quantification.info
 

 

Best regards,

Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages

 

   

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