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Gene Quantification Newsletter
March & April 2017

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  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:


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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.



highly cited paper

Guidelines for Minimum Information for Publication of Quantitative Digital PCR Experiments.
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T,
Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA.
Clin Chem 2013 59(6): 892-902

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.
recent digital PCR papers

Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.
Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, Lin M, Ying Hui L, Xu F
Biosens Bioelectron. 2017 90: 459-474

Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR.

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.
Pavsic J, Devonshire A, Blejec A, Foy CA, Van Heuverswyn F, Jones GM, Schimmel H, Žel J, Huggett JF, Redshaw N, Karczmarczyk M, Mozioğlu E, Akyürek S, Akgöz M, Milavec M.
Anal Bioanal Chem. 2017 409(10): 2601-2614

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.
Belmonte FR, Martin JL, Frescura K, Damas J, Pereira F, Tarnopolsky MA, Kaufman BA
Sci Rep. 2016 28;6: 25186

Evaluating Droplet Digital Polymerase Chain Reaction for the Quantification of Human Genomic DNA -- Lifting the Traceability Fog.
Kline MC and Duewer DL
Anal Chem. 2017 89(8): 4648-4654

Calibration-free assays on standard real-time PCR devices.
Debski PR, Gewartowski K, Bajer S, Garstecki P
Sci Rep. 2017 22;7: 44854

Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision.
Majumdar N, Wessel T, Marks J
PLoS One. 2015 10(3): e0118833

Model-Based Classification for Digital PCR -- Your Umbrella for Rain.
Jacobs BKM, Goetghebeur E, Vandesompele J, De Ganck A, Nijs N, Beckers A, Papazova N, Roosens NH, Clement L
Anal Chem. 2017 89(8): 4461-4467

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


Special Issue on digital-PCR (December 2016)
Biomolecular Detection and Quantification (10): 1-50
edited by Valerie Taly and Jim Huggett

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.

(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


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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