Advances in qPCR Europe Agenda

Registration

Coffee Break & Networking in Exhibition Hall

Digital PCR - Clinical and Translational Application
Elizabeth Day, Reseacher, MRC Laboratory of Molecular Biology, United Kingdom

I will review the basic concepts underpinning digital PCR and focus on the potential application of this powerful and versatile technique in the delivery of personalised medicine and translational medical research.

From Tissue Slide to Clinical Molecular Pathological Test Results. The Implementation of a Workflow.
Ronald Eijk, Researcher, Leiden University, Netherlands

The reliable, efficient and fast delivery of molecular pathological qPCR-test results to the clinic is challenging. We present which steps in the process can be partly or completely automated even if only very small amounts of tissue are available.

Technology Spotlight:
Droplet Digital™ PCR: a Revolutionary Approach to quantitative PCR
Pia Scheu, Application Specialist, Bio Rad Laboratories GmbH


Droplet Digital PCR (ddPCR) provides an absolute measure of target DNA molecules with unrivalled precision and sensitivity. In ddPCR, a DNA or RNA sample is partitioned into 20,000 individual nanolitre-sized droplets. After endpoint PCR is performed, the samples are read. Each droplet provides a fluorescent positive or negative signal indicating the target sequence was present or not after partitioning. The initial amount of target molecules is then quantified by counting the number of droplets with a positive amplification. ddPCR provides a revolutionary approach to target DNA or RNA quantification.

Lunch & Networking in Exhibition Hall

Poster Viewing Session

Taking Expression Profiling to New Dimensions
Mikael Kubista, Professor/Founder, TATAA Biocenter AB, Sweden

First step in new projects is usually exploratory aiming to identify interesting expression markers by screening. In classical analysis the expression of each gene is compared separately under the studied conditions and those genes that show most significant differential expression are considered important. However, variation between individuals and processing noise confounds the measured results and some of the selected genes are usually invariant of treatment but appear as false positives, while other genes that are sensitive to treatment escape notice because of the confounding variance. In my talk I will show how the precision in the selection is dramatically improved using multivariate methods that exploit correlation between genes’ expressions. The approach is particularly powerful on disintegrated samples analyzed on single cell level, since complexity is dramatically reduced. Single cell expression data are collected on high-throughput BioMark and OpenArray qPCR instrument, genes with correlated expressions are identified using GenEx, and submitted to Ingenuity database to link expression to biology. This exceedingly powerful workflow we use to study astrocytes, characterizing subtypes and changes induced under conditions such as aging and healing after induced brain damage.

Characterization of Tumor Cell Lines at Single-Cell Level
Anders Stahlberg, Senior Scientist, University of Gothenberg, Sweden

In this presentation we will discuss how single-cell analysis can be used to gain detailed information about tumor cell types, biomarkers, cell differentiation and cell functions in clonal tumor cell lines.

Coffee Break & Networking in Exhibition Hall

Drinks Reception

qPCR Analyses in the Diagnosis of Prostate Cancer
Christine Mannhalter, Professor, Medical University of Vienna, Austria

qPCR analysis of PCA3 mRNA in urine allows detection of prostate cancer cells. PCA3 mRNA levels indicate the likelihood for a positive prostate biopsy and the presence of cancer.

Development and Clinical Applications of Molecular Methods for the Detection and Molecular Characterization of Circulating Tumor Cells
Evi Lianidou, Professor, University of Athens, Greece

Molecular characterization of CTC is very important for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies

Methylation Biomarker Development and Clinical Applications and Methylation Sensitive High Resolution Melting (MS-HRM)
Tomasz Wojdacz, Post Doc, University of Aarhus, Denmark

Substantial research based evidence shows a great potential for the application of locus specific methylation changes in clinical disease management, and this talk will focus on the discovery, development and PCR technologies for the diagnostic application of methylation biomarkers.

Technology Spotlight:
Isolate. Convert. Quantify. Sigma's Complete Workflow Solution for
RT-qPCR microRNA Expression Analysis
Eric Reyes, Product Manager, Sigma Life Science

Sigma® Life Science has provided solutions for the functional analysis of microRNAs since 2008 with MISSION® microRNA mimics.  Since then, Sigma has developed more tools for understanding what genes microRNAs regulate (MISSION Target ID Library) and validating these targets (MISSION 3' UTR Lenti GoClone™).  Now, we complement the workflow at the discovery level with the MystiCq™ microRNA products for microRNA RT-qPCR analysis.

Coffee Break & Networking in Exhibition Hall

The Advantages of Targeting Genomic DNA in RT-qPCR Experiments
Henrik Laurell, Senior Staff Scientist, Inserm/Université Paul Sabatier, France

We recently developed ValidPrime, which quantifies and subtracts confounding signals derived from genomic DNA (gDNA) in RT-qPCR experiments. ValidPrime replaces the need for reverse transcriptase negative (RT-minus) reactions, and reduces the number of required control samples.

Lunch & Networking in Exhibition Hall

Poster Viewing Session

Recommandations for Precise and Robust qPCR Efficiency Estimation
Ales Tichopad, Senior Scientist , Technical University Munich, Germany

Imprecisions and robustness of determination of PCR efficiency was reviewed within controlled experiment employing simulation and recommandations were agregated based on literature reviewed, existing guidelines and our knowledge.

Improving sigmoidal fitting of real-time PCR curves
Andrej-Nikolai Spiess, Group Leader, University Hospital Hamburg Eppendorf, Germany

A major drawback of fitting sigmoidal models to real-time PCR data is the low performance in the noisy baseline region, making the extrapolation of F0 (fluorescence at cycle 0) inaccurate. We will show that modifications such as variance-weighted fitting, robust nonlinear fitting and the application of linear-quadratic-sigmoidal hybrid models can substantially increase the accuracy of deduced parameters such as F0, efficiency and threshold cycle.

Technology Spotlight:
gBlocks™ Gene Fragments—Sequence-Verified PCR Fragments
Scott Rose, Director, Integrated DNA Technologies


Integrated DNA Technologies has recently introduced a new molecular biology tool, gBlocks™ Gene Fragments. gBlocks fragments are double-stranded, sequence-verified DNA up to 500 base pairs in length. gBlocks Gene Fragments can be used as copy number standards for qPCR, without the need for cloning the endogenous amplicon. Because the average qPCR amplicon is less than 150 bp, multiple amplicons can be incorporated into a single gBlocks fragment, which is especially useful for multiplex assay controls. A single gBlocks fragment containing multiple target sequences avoids the need for linearizing plasmid controls, and ensures that concentrations of the standard dilutions are the same for the individual control assays in the multiplex reaction.

Coffee Break & Networking in Exhibition Hall

Systems-Based, Quantitative Analysis of the Regulatory Networks Underlying Cellular Differentiation
Bart Deplancke, Head, Ecole Polytechnique Federale De Lausanne, Switzerland

Presentation of our latest efforts using integrative genomics and targeted proteomics approaches as well as our gene-specific or transcript-specific qPCR primer database GETPrime to derive novel regulatory mechanisms underlying cellular differentiation.

Detecting and Resolving Position-Dependent Temperature Effects in quantitative PCR
Thomas von Kanel, Specialist for Genetic Analyses, Cantonal Hospital of Aarau , Switzerland

Positional effects due to temperature inhomogeneities of the qPCR instrument are not uncommon in qPCR. We demonstrate how such effects can be detected and resolved in order to achieve highest precision in qPCR.

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