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Session Chair: Jim Huggett, University of Surrey, United Kingdom Session Chair: Afif Abdel Nour, Universite Saint Esprit de Kaslik, Lebanon (Lebanese Republic)
Location:HS 14 ZHG -- Lecture Hall 14
Biometrology, What Is It And What Could It Mean For Biomolecular Research?
University of Surrey, United Kingdom
While biomolecular methods are widely used to measure genes and gene expression, it is well documented that such approaches are often difficult to reproduce. So while I may be able to demonstrate a two-fold difference in the expression of a given gene, it can be very difficult for you to make that same measurement. Yet, unless the reason for this discrepancy is better understood the measurement I made may be difficult to corroborated and the chance of it becoming a useful biomarker reduced. There have been several initiatives among the molecular biology community to address this, such as MIQE, and increasing efforts amongst the in vitro diagnostic community to find solutions to assist in measurement standardisation. What is perhaps less clear amongst our community is the fact that there is an entire field of science dedicated to this very problem. Metrology is the scientific study of measurement and is applied in specialties such as physics to understand the accuracy of a measurement. This is achieved by determining mathematically how it is traceable to a given unit, which at its most accurate is to the Système international d'unités, or SI. By applying the concept to biological measurements, biometrology could enable us to understand why there are discrepancies between different laboratories as well as allow us to characterise sources of error and better understand the accuracy of a given measurement. This talk will explore how this new field of research is being applied to biomolecular measurement.
Molecular Characterization By ddPCR Of In Vitro Differentiated Oocyte-like Cells From Oogonial Stem Cells
Erica Silvestris, Paola Cafforio, Stella D'Oronzo, Claudia Felici, Francesco Silvestris, Giuseppe Loverro
University of Bari Aldo Moro, Italy
Recent reports regarding the presence of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, there isn’t consensus on isolation methods of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes.
Ovarian cortex fragments from menopausal and non-menopausal women were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and cultured for up to 3 weeks. Large and small cells were individually isolated by DEPArray technology and early and late differentiation markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content was investigated using fluorescence in situ hybridization (FISH).
After immuno-magnetic enrichment, DDX4-positive OSCs from non-menopausal and menopausal women, under appropriate culture conditions, differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. Moreover, in culture small DDX4 positive cells are also present which do not express differentiation markers.
Therefore, we provide further evidence demonstrating the presence of stem-like cells with ovarian germ line properties within the otherwise exhausted oocyte reserve of menopausal human ovaries. These cells can be a source of oocytes that can be exploited to achieve fertility in women who are infertile or have an exhausted ovarian reserve.
Higher Order Multiplexing Using Digital PCR for CRISPR Gene Edit Validation
Alexandra Sarah Whale
LGC, United Kingdom
'Higher order multiplexing’ is the unique ability of digital PCR (dPCR) to precisely measure more targets than there are detection channels in the same reaction. In quantitative PCR, in order to measure three targets, three detection channels are required, however, in dPCR, the partitioning of the reaction into discrete partitions prior to the PCR enables three or more targets to be detected with two detection channels. This is achieved by varying the amount of primers and/or probes added to the reaction so that the end-point fluorescence is different between two targets with the same detection probe; the end-point fluorescence generates different “clusters” of partitions that can be observed on a 2D scatter plot. This talk will describe the different multiplex assay design strategies we have developed for genotyping and how precise and sensitive quantification can be achieved from counting the number of partitions in each of the clusters. The main example will illustrate our assay design for genotyping knock-in and knock-out CRISPR gene edits using a combined tandem probe binding and drop off probe design that we use to validate and determine the efficiency of desired gene edit.
Serial Flow Digital Droplet PCR, a New Paradigm!
Dropworks, Inc., United States of America
DropWorks introduces FluxPCR™; a fast and flexible platform for performing digital assays in self-contained flowing droplets. Aiming to help users transition from qPCR to digital PCR, DropWorks has reduced both the cost and complexity required to generate high quality digital PCR data. A single touchpoint workflow reduces error and serial sample processing provides the flexibility to run only a few samples or an entire plate without wasting expensive consumables. An instinctive user interface and software streamlines data production and ensures that users can spend less time processing their data and more time understanding what that data means for the underlying science.
Eight Years Later This Is What Digital PCR Offered To Science
Afif Abdel Nour
The Holy Spirit University of Kaslik, Lebanon
Throughout the past 36 years Polymerase Chain Reaction (PCR) has proven to be the most powerful technique in a scientist toolbox. The overwhelming evolution from the first generation (PCR) to the “Gold standard” second generation (Real Time quantitative PCR A.K.A. qPCR) and recently to the third and breathtaking generation the digital PCR (dPCR), proven that DNA/RNA amplification will always be a popular method.
To understand the use of digital PCR, the Scopus database was screened for the terms “digital PCR, ddPCR and dPCR”, in all papers that were published between 2011 and 2018. We considered only papers published in journals and we excluded books, books chapters and conferences. Then we classified those papers by country, subject areas, CiteScore, institutions... The aim of the current analysis is to shed the light on the good and bad students in terms of usage of the dPCR and the citation of the digital MIQE guideline. We than studied the impact of dPCR on different research area like oncology, single cell analysis and diagnostics.
Highly Multiplexed Detection of GMOs in Food Samples by Crystal Digital PCR
Katharina Lührig1, Caroline Charky2, Maximilian Neugebauer1, Heike Ziebarth1, Kornelia Berghof-Jäger1
1BIOTECON Diagnostics GmbH, Germany; 2Stilla Technologies, France
Stilla Technologies will present the latest technical updates of the NAICA system along with the outcome of a cooperation between Stilla Technologies and BIOTECON Diagnostics to deliver a complete solution for the detection of soy GMOs in food samples.
As the number of authorized GMOs in the European market is increasing, faster and cost-efficient detection methods are needed. We present digital PCR as a suitable alternative to real-time PCR which allows multiplexed GMO quantification without the need for a standard curve. A new digital PCR assay developed by BIOTECON Diagnostics on the NAICA system allows the quantification of all 14 soybean GMO events currently authorized in the EU in just a single reaction. This assay reliably and precisely quantifies GMO contents at the regulatory thresholds of 0.9% and 0.1%. A summary quantification of all authorized soy events present in a sample is provided making quantification with single assays dispensable. We will highlight the particular advantages of the Crystal Digital PCR platform for R&D and routine analytics.
Resolving Translational Oncology Research Challenges with NanoString Direct Digital Gene Expression Analysis.
Nanostring Technologies, United Kingdom
Successful Translational Research and Biomarker development today much depends on the availability of technologies that ensure accurate and precise multiplexed detection and quantitation of target biomolecules. Clinical necessities, on the other side, dictate that such measurements can be done effectively on small sample input amounts, on material of poor starting quality, and analytes obtained in a minimally invasive way. Because of this, classical methods often encounter problems to measure RNA or Protein expression levels robustly and reproducibly at high multiplexing levels.
The NanoString digital gene expression technology overcomes these hurdles by offering a fast workflow that is just based on hybridization and does not require any enzymatic modification of analytes or library preparation. The talk will present achievements of the NanoString approach in the context of biomarker discovery and pathway analysis within various areas of cancer research. It will also show how the technology can be employed to resolve expression differences in tissue sections in the spatial dimension.