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|Date: Tuesday, 19/Mar/2019|
|8:30am - 12:30pm||dPCR: digital PCR|
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Afif Abdel Nour, Universite Saint Esprit de Kaslik, Lebanon (Lebanese Republic)
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
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
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
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
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.
|8:30am - 12:30pm||LB: Liquid Biopsy|
Session Chair: Jo Vandesompele, Ghent University & Biogazelle, Belgium
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Cell-Free Tumor DNA Analysis In Liquid Biopsy Using Ultrasensitive Sequencing
1University of Gothenburg, Sweden; 2Sahlgrenska University Hospital, Sweden
Liquid biopsy and detection of tumor associated-mutations in cell-free circulating DNA (ctDNA) often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage, polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. We show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. Theoretically, high fidelity enzymes will reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.
Clinical Application of Liquid Profiling for Precision Medicine
German Heart Center Munich, Germany
The concept of “precision medicine” in the treatment of cancer patients aims to specifically target deregulated molecular cancer pathways that are involved in cancer cell proliferation, angiogenesis, metastasis and evasion of the immune control. Classical ways are extracellular blockage of membrane-bound growth receptors by specific antibodies or intracellular inhibition of cancer pathways by small molecules like tyrosine kinase inhibitors that have shown promising therapeutic results. New approaches aim to restore and reactivate immune cell functions to attack the tumor in a more sustainable way. Precondition for precisely acting drugs is the presence of the respective molecular changes that have to be detected in tumor tissue or in the blood plasma – also known as “liquid biopsy” or “liquid profiling” – prior to therapy.
Sensitive blood-based diagnostics are able to identify druggable mutations in cell-free plasma tumor DNA (ctDNA) and circulating tumor cells (CTC). Current diagnostic strategies include single-, multi-gene and whole exome / genome approaches that have recently become more sensitive and specific by the introduction of tumor-enrichment and error-reducing techniques. As liquid profiling is only minimally invasive it can be used to complement tissue biopsy for patient stratification and for the serial monitoring of successfully treated and newly occurring resistant cell clones at an individual level.
With only few exceptions, plasma ctDNA is measurable in patients with most tumor types, particularly at advanced stage of disease. Concordance with tumor tissue is around 90% if highly sensitive methods are used. ctDNA diagnostics support therapy stratification if tumor biopsy is not available or insufficient, has predictive and prognostic power and can be used as modern, quantifiable tumor marker for monitoring mutation-positive patients. Finally, multiplexing and sequencing enables the detection of new mutations. Preanalytics and rigorous quality control have turned out to be critical for reproducible results. Today, the use of blood conserving tubes, double centrifugation, standardized DNA extraction, enrichment, quantification and sequencing techniques are basic requirements for ctDNA diagnostics.
Plasma-based ctDNA “companion diagnostics” has developed to a valuable new tool for precision medicine. First assays are available as IVD-CE labelled methods to be applied in routine diagnostics. High grades of technological and quality standards as well as future combination with protein and metabolome markers will help to improve the diagnostic accuracy and facilitate new applications in other fields of precision medicine such as in immune checkpoint therapies.
Exploiting RNA in Liquid Biopsies for Precision Medicine Purposes
1Ghent University, Belgium; 2Biogazelle, Belgium
In contrast to general belief, a substantial part of the human transcriptome is abundantly present in the blood and other biofluids as extracellular messenger RNA, long non-coding RNAs and various small RNAs, ready to exploited. I will discuss various workflows for RNA sequencing of biofluid derived RNA, including probe-based target capture and unbiased total RNA library prep as sensitive RNA sequencing workflow to study thousands of mRNA and lncRNA genes in cell-free RNA from patients’ plasma and other biofluids. Apart from RNA abundance profiling, this type of data can also be used to detect structural RNA variants, such as somatic mutations, fusion genes and RNA editing events, all known to play an important role in disease, including cancer. The resulting RNA profiles can be deconvoluted to enumerate the cells, tissues and organs that contribute to the extracellular RNA. Human biofluid RNA sequencing enables liquid biopsy guided precision oncology, such as therapy stratification, treatment response monitoring and early detection of relapse. I will also discuss the pre-analytical jungle of RNA targeted liquid biopsies and need for standardization, as part of the ongoing extracellular RNA quality control study. I will end with the first insights of the Human Biofluid RNA Atlas, in which we have deeply probed into the extracellular transcriptome of 22 human biofluids, providing a solid foundation for exploiting biofluids for diagnostic purposes. (on behalf of Extracellular RNA Quality Control consortium, Human Biofluid RNA Atlas consortium).
Liquid Biopsy -- Exosomal microRNA Biomarker Signatures in Clinical Diagnostics.
Division of Animal Physiology & Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany
Extracellular vesicles (EVs) circulate in body liquids and are involved in the intercellular communication. They have important regulative functions in almost any physiological or pathological process. In recent time especially the exosomes have gained huge scientific interest because of their molecular diagnostic potential, mainly based on the containing microRNA pattern. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs and exosomes, primarily from blood compartments. The exosome purity and which subpopulations of EVs are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the overall performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill, mainly pneumonia and sepsis patients. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To analyze the small-RNA deep sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (via isomiRROR) was applied.
Final goal was the development of microRNA/isomiR biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various patient cohorts were investigated: healthy volunteers, sepsis (referred to mild or severe pneumonia), acute pulmonary failure (ARDS) and septic shock. Distinct miRNA signatures were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS or sepsis shock. The study results indicate that EV miRNA biomarkers have future potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Further the methodological findings provides guidance for navigating the multitude of EV/exosome isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy.
New Developments in Sample Quality Control - Focus on Cell-free DNA
Agilent Technologies, Germany
Quality control of nucleic acid starting material is essential to ensure the success of downstream experiments. Especially, Next Generation Sequencing (NGS) developed to a powerful tool in almost all genetic research and diagnostic areas. Due to the establishment of low input library protocols for NGS workflows sequencing of cell-free DNA (cfDNA) became possible. Since the downstream applications are often time-consuming and expensive, tight QC steps are required to avoid a “garbage in-garbage out” situation.
This talk focuses on standardized nucleic acid quality assessment using different automated electrophoresis platforms to ensure that samples are “fit for purpose”. Accurate quantification of starting material (DNA, RNA, cfDNA, FFPE samples) is essential to determine suitable input amounts for library preparation prior to sequencing. Depending on preanalytical sample treatment or extraction methods the quality of e.g. cfDNA can vary. This results in various electropherogram patterns after electrophoretic separation as presented in this talk. Moreover, the electrophoretic separation enables to qualify cfDNA samples according to their contamination level with high molecular weight material. Likewise, quality scores for gDNA, RNA as well as FFPE RNA can be assessed using automated electrophoresis systems, which allow defining a quality threshold for specific types of samples or preparation. This allows defining thresholds for objective sample qualification prior to library preparation.
Exosome-derived Epigenetic Biomarkers for Saliva Diagnostics
1AIT Austrian Institute of Technology GmbH, Molecular Diagnostics, Vienna, Austria; 2Medical University Vienna, Division for Endocrinology and Metabolism, Vienna, Austria
The aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis and prognosis. To this end we have been establishing and optimizing a whole range of multiplex capability technologies (e.g. microarrays, quantitative PCR, Luminex bead technology) to meet the special demands and challenges of diagnostic biomarker discovery - and validation in body fluids. Using this specific technology expertise we e.g. successfully discovered autoantibody- as well as DNA methylation -based diagnostic marker panels for the big 4 cancer entities (breast, colon, prostate, lung) in serum or plasma. Based on these success stories and the evident advantages of saliva as a diagnostic matrix our recent special interest is to go for saliva diagnostics and to evaluate saliva for its suitability for circulating biomarker-based diagnostics. Along these lines we will show proof of concept studies for autoantibody- and DNA-methylation based salivary diagnostics and report on the evaluation of different commercially available strategies for isolation of exosomes from human serum and saliva. We will further present data from comparative profiling studies in salivary - and serum-derived exosomes including targeted protein-, genome-wide microRNA – as well as DNA-methylation profiling. Last but not least we will report on first results of a research project where we are looking for salivary and plasma exosome-derived epigenetic biomarkers for early type 2 diabetes diagnosis.
Sensitive, Easy And Fast Monitoring Of Treatment Efficiency, Resistance Development And Disease Progression Using Open Platform QPCR On Liquid Biopsies From Cancer Patients
Personalized administration of present and future targeted cancer therapies demands fast, low cost and easy-to-use companion diagnostics (CDx) providing doctors and patients with the answers they need to maximize treatment efficiency and minimize treatment costs. We have developed and clinically validated several real-time PCR based assay for liquid biopsies. Sensitivity studies show that our SensiScreen® Liquid assays can detect down to a single copy of mutated DNA in a background of wild type plasma cell-free DNA (cfDNA) in a simple and robust workflow.
The extreme sensitivity of our assays is obtained by use of BaseBlockers™, that suppress amplification of wild type DNA while allowing amplification of mutated DNA, modified primers and sensitive dual-labelled Probes. BaseBlockers™, primers and probes are all based on the DNA platform technology – Intercalating Nucleic Acid, INA®.
We will present a case study using our SensiScreen® Liquid CE IVD Assay for monitoring disease progression and treatment efficiency in a clinical setting, in a BRAF V600E mutated metastatic colorectal cancer patient. After initial diagnosis and genotyping, we have monitored the patient’s cfDNA levels and the cfDNA amount of BRAF V600E in plasma for more than 700 days during three different regimes of treatment and compared with carcinoembryonic antigen and CA 19-9 levels.
We believe that sensitive monitoring of liquid-based biopsies present unique opportunities for healthcare providers, targeted therapy developers, and eventually cancer patients. The information and additional knowledge gained on patient response and efficacy of treatment can be used as a tool to reduce use of inefficient treatment and improve clinical output.
|12:30pm - 2:00pm||Journal: BDQ Editors Meeting|
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
|12:30pm - 2:00pm||PO2: Tuesday Lunch Poster Session|
All posters will be displayed in parallel at all three poster sessions PO1, PO2 & PO3 => the poster viewing can be done from "Monday Evening Main Poster Session" till "Wednesday Lunch Poster Session"
|12:30pm - 2:00pm||LU2: Lunch Tuesday|
|2:00pm - 6:00pm||AMDx1: Advanced Molecular Diagnostics 1|
Session Chair: Carl T Wittwer, University of Utah, United States of America
Session Chair: Mikael Kubista, TATAA Biocenter AB, Sweden
Rapid qPCR and the Future of Molecular Diagnostics
University of Utah, United States of America
We experimentally dissected the kinetic requirements of PCR into the stages of DNA denaturation, primer annealing, and polymerase extension. A commercial capillary LightCycler™ was used to correlate PCR product length to required extension times. More accurate results for all stages were obtained by using a custom “extreme” temperature cycling instrument. Increased primer and polymerase concentrations were allowed to increase speed, and actual temperatures were measured in real time rather than programmed or predicted. Two stages were left long and not limiting, while the other was varied until Cqs increased. Under the conditions used, polymerase extension times depended on product length with about 1 s needed for every 100 bps. Cqs started to increase after 200-500 ms above the denaturation threshold, and after 300-1,000 ms below the annealing threshold. Temperature thresholds were set by including >98% of the derivative melting curve areas as experimentally determined by melting curves under PCR conditions. Progressing from rapid cycle PCR to extreme PCR has decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.
Impact of Endogenous and Exogenous Alterations of Glucocorticoids on the Derangement of Clock-related Genes in Peripheral Mononuclear Cells
Sapienza University of Rome, Italy
Circadian clock genes regulate the physiological sensitivity to rhythmic release of glucocorticoids (GCs). In turn, GCs have reciprocal effects on circadian system. Conditions affected by hyper- or hypocortisolism are characterized by a loss of circadian rhythmicity. Loss or impairment of circadian rhythms have been reportedly associated with malignancies and an increased incidence of infectious diseases, suggesting a role of immune function alterations. Adrenal insufficiency (AI) requires life-long glucocorticoid replacement. Conventional therapies fail to mimic the endogenous cortisol circadian rhythm. Objective of this study was to evaluate the effect of the timing of GC administration on circadian gene expression in peripheral blood mononuclear cells (PBMCs) of AI patients from DREAM trial. Patients were randomly assigned to continue their multiple daily doses, or switch to an equivalent dose of once-daily modified-release hydrocortisone, and compared to healthy controls. Circadian genes in PBMC were analyzed by quantitative by real-time qRT-PCR using predesigned 96-well panel SYBR® Green Circadian rhythms (SAB Target List) H96 (Bio-Rad, PrimePCR®). Compared to healthy controls, 19 of the 68 genes were found differentially expressed in AI patients at baseline, 18 of which were restored to control levels 12 week after switching therapy, comprising: ARNTL, CLOCK, PER3, TIMELESS, AANAT, CAMK2D, CREB1, CREB3, MAPK1, WEE1, PRF1. Changes in WEE1, PRF1 and PER3 expression correlated with change in clinical outcomes including glycated hemoglobin, inflammatory monocytes and CD16+ NK cells, suggesting that the extent of reprogramming of circadian gene expression can be linked to the magnitude of improvement in clinically measurable outcomes. In conclusionAIpatients on standard therapy exhibit a dysregulation of circadian genes in PBMCs. The once-daily administration reconditions peripheral tissue gene expression to levels close to healthy controls, and correlates with clinical improvement.
SD DNA Polymerase: A New Tool for Variety of Molecular Biology Applications
Till recently DNA polymerases were either suitable for PCR amplification or isothermal amplification, but not both. The new SD polymerase (Bioron GmbH Patent US 9,896,671) is a Taq DNA polymerase mutant that was created as a result of successful attempt to combine the thermostability and robust polymerase activity of Taq DNA polymerase with strong strand displacement activity of Bst DNA polymerase. Currently SD polymerase seems to be the only enzyme suitable for PCR which possesses strong strand-displacement activity. New polymerase was shown to be suitable in the number of unique applications such as heat pre-denaturated LAMP and PCDR (Polymerase Strand Displacement Reaction). SD polymerase appeared to be effective in conventional PCR, Long-PCR and amplification of GC-regions with complex secondary structures. Moreover, SD polymerase can be effectively used in WGA (Whole Genome Amplification) based on DOP-PCR, in the Single Cells WGA, in the new NGS-library construction methods and in the newly developed template-independent tailed tandem repeat PCDR (TTR-PCDR).
Since SD-polymerase allows performing Strand Displacement and PCR in the same reaction, the work flow for the SD polymerase based techniques are usually comfortable, simple and friendly.
New techniques based on the advantageous properties of SD polymerase are currently under development by several biotech companies over the world. Thus, SD polymerase is able to improve the existing DNA amplification techniques and can be used for creation of new convenient methods of DNA manipulations.
Methylation Sensitive High Resolution Melting; A Sensitive And Specific Method For High-Throughput Assessment Of Methylation.
IRCCS Fondazione Don Carlo Gnocchi, Italy
DNA methylation is an epigenetic mechanism which implies heritable changes of gene expression without a change in the primary DNA sequence. Covalent histone modifications and methylation changes of cytosine at CpG dinucleotides are the most widely investigated epigenetic mechanisms. DNA regions with a relatively high CpG dinucleotide content are referred to as CpG islands that are distributed in a non-random manner across the human genome and often span 5’ untranslated region (UTR), 3’ UTR, promoter region and the first exon of protein coding genes. Methylation of CpG islands usually acts to turn off (silence) transcription by recruiting histone deacetylases thereby inducing the formation of inactive chromatin. Mapping of methylation patterns in CpG sites is an important tool for understanding both normal and pathogenic gene expression events.
Numerous technique are used for detection of CpG methylation, among them, PCR-based protocols are most widely used. Because PCR amplification removes methylation marks, the DNA template is chemically modified by sodium bisulfate that converts all unmethylated cytosines to uracil, leaving methylated cytosines unaltered and preserving methylation information before PCR amplification.
Subsequent amplification of bisulfite-modified template results in different amplicons from methylated and unmethylated templates with different melting profiles when subjected to thermal denaturation.
The methylation-sensitive high resolution melting (MS-HRM) technology is based on the comparison of the melting profiles of sequences that differ in base nucleotide composition. The PCR product originating from the methylated allele will have different GC content from PCR product derived from unmethylated variant of the same locus. MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio (range from 0 to 100 methylation percentage).
Here, we show the application of MS-HRM in two different studies:
1) detection of methylation levels in the 3’UTR of dystrophia myotonica protein kinase (DMPK) gene in a cohort of 66 myotonic dystrophy type 1 (DM1) patients (age 38.6±12.5 years) and 30 age-matched healthy controls (age 40.3±13.8 years).
2) detection of promoter methylation levels in cannabinoid type 1 (CNR1) and 2 (CNR2) receptors of 12 multiple sclerosis secondary progressive (MSS-SP) patients (age 54.2±11.7 years) before and after treatment with Sativex®.
Both papers show how the MS-HRM protocol provides a high-throughput platform for cost- and labor-efficient screening for methylation changes. Moreover, the simplicity and high reproducibility of this technique makes MS-HRM the method of choice for methylation assessment in both research and diagnostic applications.
Two-Tailed Quantitative PCR for Precison Diagnostics
1TATAA Biocenter AB, Sweden; 2Institute of Biotechnology, Czech Academy of Sciences
We present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. The reverse transcription step can be multiplexed and it allows for rapid testing with a total analysis time of less than 2.5 hours. Several applications will be presented including a quality control panel for liquid biopsy samples and 1-tube combined analysis of mRNA and microRNA.
P. Androvic, L. Valihrach, J. Elling, R. Sjöback, M. Kubista. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages e144
Precision Liquid Biopsy Based Nucleic Acid Based Molecular Diagnostics Powered by Xenonucleic Acids
DiaCarta, Inc., United States of America
Current clinically available molecular tests for detection of pathogenic nucleic acid variations especially tumor derived oncogenic 'driver' and drug resistant somatic mutations that are performed on circulating cell-free nucleic acids present in biological fluids such as patient’s blood plasma have limited sensitivity. This is because of the low frequency of these gene variations and the large excess of wild-type nucleic acids present. In order to achieve high sensitivity for the detection of only a few target molecules (mutant alleles) present in a vast excess of non-target molecules (wild-type alleles) sophisticated methodologies that require expensive instrumentation, highly skilled operators and in some cases intensive computational bioinformatics methods such as digital-droplet PCR (ddPCR), BEAMing PCR and next generation deep sequencing (NGS) are being employed in large clinical research centers. The limited availability, high cost and long analysis times of these methods prompted us to develop a new technology that can be performed globally by existing pathology personnel with instrumentation that is already present in every hospital pathology laboratory. At the heart of this innovative technology are novel molecular nucleic acid analogs that we call xenonucleic acids (XNA) that possess all the natural bases that occur in DNA appended to a new chemical backbone that imbibes these oligomeric nucleic acid binding molecules with exquisite specificity and high binding affinity for complementary target sequences. Any variation in the sequence that the XNA binds to creates a differential binding phenomena that can be exploited to develop real-time qPCR and extremely high sensitivity NGS assays that can detect as little as 2 copies of variant templates in a large excess of wild-type templates in DNA obtained from tissue biopsies or more preferably plasma. Commercial CE/IVD Certified Products have been developed and validated that include QClampTM gene specific real-time qPCR based tests, a new highly sensitive blood-based colorectal cancer detection test called ColoScapeTM and a high sensitivity targeted amplicon based target NGS platform called OptiSeqTM. This presentation will discuss the new technology and the improved and widely available opportunities that it affords for improved precision diagnostics and targeted therapies of human diseases particularly cancer.
Multiplex xMAP Technology for a Complex Detection of Pathogenic Agents Significant for the Protection of Human and Animal Health
Veterinary research institute/Brno, Czech Republic
Early detection of pathogenic agents in the human and veterinary field is one of the main and key moments for the treatment of infectious diseases. For this we use molecular biological methods, such as a polymerase chain reaction (PCR). Methods of molecular diagnostics are generally focused on finding specific or virulence genes and therefore the subsequent design of probes which are necessary for detection of the selected pathogen. Also specific mutation in gene, or single nucleotide polymorphism (SNP) can be used in probe design. Nowadays, a large number of pathogens need to be identified during one reaction in a short time. One of the possible multiplex sample analysis is xMAP technology (x = analyte, MAP = Multi Analyte Profiling). xMAP technology is based on a combination of existing laboratory methods such as PCR, flow cytometry and ELISA, and enables detection of more than 50 different analytes (nucleic acids) simultaneously during one reaction. In this case, a multiplex oligonucleotide ligation (MOL) is performed prior to the PCR in which there is only one pair of universal primers. One of the primers is labeled with a fluorescent dye. xMAP technology uses magnetic microspheres with a special spectral address, to which the analyte is then binds specifically with the sample.
In our laboratory, we focused on obtaining a comprehensive protocol for the MOL-PCR method followed by MagPix analysis. The assay is suitable for rapid multiplex detection of bacteria, parasites and also viruses in real samples. The final result of this work is the creation of individual multiplexing systems (detection panels). At present, we have developed diagnostic panels for the multiplex detection of 5 bacteria (Campylobacter jejuni, Escherichia coli – EHEC, Yersinia enterocolitica, Listeria monocytogenes, Salmonella enterica) and 4 parasites (Toxoplasma gondii, Taenia saginata, Trichinella spiralis, Giardia intestinalis). We continue to develop a panel for identification of pathogens that can be used in bioterrorism: Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis.
This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.
|2:00pm - 6:00pm||ISEV: ISEV Satellite Session|
Session Chair: Kenneth Witwer, The Johns Hopkins University School of Medicine, United States of America
Session Chair: Hidetoshi Tahara, Hiroshima University, Japan
Mesenchymal Stem Cell-derived Extracellular Vesicles: a Potential New Tool in Regenerative Medicine
Institute for Transfusion Medicine, Germany
Human mesenchymal stem/stromal cells (MSCs) represent a promising tool in regenerative medicine. Up to now, more than 800 NIH-registered clinical trials investigated their immunomodulatory and pro-regenerative therapeutic potential in various diseases, including graft-versus-host disease (GvHD) and ischemic stroke. Despite controversial reports regarding the efficacy of MSC-treatments, MSCs seem to exert their beneficial effects rather in a paracrine manner than by cell replacement. In this context, extracellular vesicles (EVs), such as exosomes and microvesicles, are discussed to execute the MSCs’ therapeutic effects. Indeed, we observed beneficial therapeutic impacts of MSC-EVs in a patient, who suffered from steroid-refractory acute GvHD. Furthermore, beneficial effects were observed in animal models for several different diseases.
According to controversial reports in the MSC field, especially since a phase III clinical trial failed to show clinical efficacy in MSC treated GvHD patients, we have started to compare immunomodulatory effects of independent MSC-EV preparations. Indeed, in our in vitro assays independent MSC-EV fractions reveal different immunomodulatory capabilities. To unravel the basis for these differences we are currently using several methods to dissect the heterogeneity between and within given MSC-EV samples.
Standardization in EV and exRNA Research: MISEV and Beyond
The Johns Hopkins University School of Medicine, United States of America
Rapidly growing interests in physiologic and pathologic processes associated with extracellular vesicles (EVs), including presumed functions of various EV RNA molecules, have created a need for standardization initiatives to shape and guide the field. As an example, the International Society for Extracellular Vesicles (ISEV) first published the Minimal Information for Studies of Extracellular Vesicles (“MISEV”) in 2014 to call attention to rigorous EV characterization approaches and experimental controls. In this presentation, I review recent developments in EV and exRNA standardization, focusing especially on the “MISEV2018” guidelines in the Journal of Extracellular Vesicles. These guidelines were prepared with methods for broad community input and buy-in that may serve as a model for ongoing and future initiatives. We will discuss outstanding needs in the field and what might be done to address them.
Circular RNAs In EVs - A Novel Class Of Released Non-coding RNAs
Justus Liebig University of Giessen, Institute of Biochemistry
Non-coding RNAs (ncRNAs) were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs), which represent the newest class of ncRNAs, turned into research focus within the last decade. Although single examples of these particular RNA class had been known for more than forty years, circRNAs were established as a large RNA class only a few years ago, based on the advent of tailored RNA-Seq technologies and Bioinformatics. CircRNAs were identified in all eukaryotic cells investigated so far, are cell-type specifically expressed, and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form, resulting in a metabolically more stable form of RNA compared to their linear counterparts. These biological properties and the correlation with various human diseases, such as cardiovascular diseases and cancer, immediately suggesting their potential use as novel attractive RNA-based biomarkers.
We have focused on the analysis of circRNAs and the corresponding linear splice isoforms from human blood cells, in particular platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles (EVs) from purified and in vitro activated human platelets, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. Furthermore, to unravel mechanisms that contribute to the specific packaging of RNAs into EVs, we developed a novel approach to determine specific sequence motifs required for selective loading of (circ)RNA into EVs. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs.
In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and harbor great biomarker potential. In addition, we would also like to stress the strong need for standardization and for setting up minimal requirements for validating this class of ncRNA.
Characterization of Extracellular vesicles (EVs) Derived ExRNA in Cancer Using NGS
1Department of Cellular and Molecular Biology,Institute of Biomedical & Health Sciences, Hiroshima university, Japan; 2Department of Head and Neck Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan.
MicroRNAs (miRNAs) are small non-coding RNAs approximately 21 nucleotides in length that regulate posttranscriptional gene expression. miRNAs exist in exosomes, which are 50–100nm in size extracellular vesicles (EVs) and secrete from mammalian cells. Recent studies have demonstrated that miRNAs and other small RNAs stably exist in body fluids and their expression patterns in cancer patients are distinct from those in healthy individuals. In this study, we analyzed non-coding small RNAs including miRNAs that specifically exist in plasma/serum of patients with breast and tongue cancer by using NGS plat form. We identified a number of biomarker candidates such as mature miRNAs, isomiR, tRNA-derived fragments (tRFs), and other ncRNA, are known to regulate expression of genes involved in cell metabolism and are released into body fluid from various cells with extracellular vesicles. To test the possibilities that these candidate small RNAs are secreted from cancer cells, we purified EVs from serum in health volunteer and cancer patients, and analyzed small RNAs using NGS, and found that some of candidate small RNAs can be found in EVs. Interestingly, these small RNAs are also found in culture media in cancer cell lines. Based on our alogism, we identified significant biomarker secreted from cancer cells for early detection of cancer.
Distribution Of Non-coding RNA Types Over EV And Other RNA Carriers In Plasma
Utrecht University, The Netherlands
EV contain RNAs, lipids, and proteins and act as multicomponent signaling entities during intercellular communication. The release and composition of EV depends on the activation/differentiation status of EV-producing cells.
The currently most intensely studied EV-RNA biotypes are miRNAs and mRNAs, some of which have been implicated in disease progression and/or proved valuable as biomarkers. However, we previously showed that EV released by many different cell types are particularly rich in other small RNA biotypes such as tRNA, Y-RNA, snRNA, and snoRNA, which may also exert gene regulatory functions. Examples will be shown on how environmental triggers can lead to changes in the type of released EV and their small non-coding RNA content. Levels of not only miRNAs, but also other non-coding RNA types varied depending on the status of the parent cell and these may be further explored as biomarkers or functional entities within EV.
RNA in plasma is not only enclosed in EV but can also be associated to other macromolecular structures, such as ribonucleoprotein particles (RNPs) and lipoprotein particles. These structures overlap in size and/or density with EV and may be co-isolated in commonly used EV isolation procedures, such as ultracentrifugation. By using several separation techniques and protease/RNase treatment strategies, we show how different miRNAs and other non-coding RNAs are distributed over various carrier structures in plasma. In-depth characterization of these RNA carriers and changes in their RNA content induced by cell activation or disease is important to further elucidate the function and biomarker potential of the different extracellular RNA types.
Normalizing Urinary Extracellular Messenger RNA Biomarkers: Theoretical Considerations and a Review of Experimental Findings
1University of Michigan Medical School, United States of America; 2Vanderbilt University School of Medicine, United States of America
Messenger RNA (mRNA) has been extensively annotated, and its crucial role in the central dogma has made it a key target in many studies of biomarkers and drug targets. Extracellular vesicles shuttle mRNA, among other molecular cargo. mRNA in urinary extracellular vesicles has potential as a biomarker, particularly in diseases affecting cells of the urothelial tract. There is evidence that this mRNA could provide information about transcription in cells of urogenital tissues. However, the optimal means of normalizing these signals is unclear. In the more common cell lysate context, gene expression can be normalized to robustly expressed genes that have similar expression between cells. However, in the context of extracellular mRNA shuttled by extracellular vesicles, normalization strategies remain undefined.
This talk will describe relevant first principles as well as research findings from our lab and other labs toward normalization of urine extracellular mRNA. The talk will focus on at least two hypothetical sources of confounding that might be important denominators when normalizing assays of urinary extracellular mRNA transcripts. The first is changes in the composition of the biofluid matrix broadly affecting analytes’ concentrations (i.e., intra-individual changes or inter-individual differences in urine composition due to having recently consumed water). The second is broad changes or differences in the expression of genes without direct relevance to the specific biology of interest. The extent to which each of these factors must be taken into account during normalization will depend on the magnitude of target gene’s expression signal in the disease relative to the magnitude of the noise created by these potentially confounding factors. The rationale for the use of urinary creatinine as the traditional reference molecule for many urinary assays will be discussed, as will advantages and disadvantages of urinary creatinine for normalization of urinary extracellular mRNA. In addition, the possibility of using reference genes to normalize assays of urinary extracellular mRNA will be discussed. Prior experiments bearing on the feasibility of these approaches will be reviewed. The talk will also discuss other potential strategies for normalization, such as normalizing to the concentration of extracellular vesicles. Finally, the talk will attempt to synthesize these ideas and the findings from the literature into suggestions for those developing urinary extracellular mRNA assays.
Serum-Free Media Supplements Carry MiRNAs That Co-Purify With Extracellular Vesicles
Johannes Gutenberg University of Mainz, Germany
Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing fetal bovine serum (FBS) or a defined media supplement as nutrient. Recently, Wei et al. (2016, PMID: 27503761) reported that miRNAs are co-isolating with EVs when harvested in media supplemented with FBS or vesicle-depleted FBS. To avoid serum-associated miRNA contamination, we performed RNA-Seq of EV-associated miRNAs derived from primary oligodendrocytes cultured under serum-free conditions and subsequently validated miRNAs by RT-qPCR including media and supplement controls. Intriguingly, several EV-associated miRNAs were robustly detected in un-conditioned media subjected to the EV-isolation protocol and the media supplements NS21 and B27, which are routinely used for neural cell culture. RNAse and detergent treatment of NS21 removed most but not all of the contaminating miRNAs. Detailed analysis of NS21-supplement by screening individual components excluded BSA as major source of the miRNA contamination and identified a single component as carrier of miRNAs. Media supplement lacking the miRNA-carrying components however appears to affect cell viability, indicating that deprival of this specific supplement is not a solution to get rid of contaminating miRNAs. Based on these findings, we designed a new RNA-Seq strategy including EV-samples collected under conditions of supplementation or supplement-deprivation as well as supplement only control.
Taken together, our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV-samples. Consequently, analysis of EV-RNA needs refined strategies including respective media controls.
|7:00pm - 11:59pm||DINE: Event Dinner @ Bräustüberl|