Logo qPCR dPCR & NGS 2017

Conference Agenda

Overview and details of the sessions of this conference. Please select a date or location to show only sessions at that day or location. Please select a single session for detailed view (with abstracts and downloads if available).

Session Overview
ncRNA: non-coding RNAs
Tuesday, 04/Apr/2017:
2:00pm - 6:00pm

Session Chair: Pieter Mestdagh, Ghent University, Belgium
Session Chair: Michael W Pfaffl, TUM, Germany
Location: Lecture hall 15
350 participants, TUM Weihenstephan

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Cancer-specific long non-coding RNAs as novel biomarkers and targets for therapy.

Pieter Mestdagh

Ghent University, Belgium

Until recently, it was believed that only a small fraction of the genome contained relevant information, used by the cell to produce proteins. The majority was referred to as ‘junk DNA’ with no obvious function throughout life. The introduction of massively parallel RNA-sequencing technology has drastically changed that view. Today, there’s ample evidence demonstrating that the majority of the genome is transcribed, producing non-coding RNA (ncRNA) transcripts that differ in size, shape, expression and function. The bulk of the non-coding transcriptome consists of so-called long non-coding RNAs (lncRNAs). These lncRNAs are characterized by an exquisite tissue-specificity of lncRNAs which makes them extremely attractive as targets for therapeutic intervention or biomarkers for disease diagnosis and treatment response monitoring.

The potentials of isomiRs -- a new dimension in biomarker research.

Benedikt Kirchner, Ming Wu, Dominik Buschmann, Michael W. Pfaffl

Animal Physiology & Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany

Recent studies have proven that microvesicles are a valuable source of biomarkers in a number of physiological and pathophysiological states. The molecular content of exosomes (and other vesicles) and their miRNA cargo in particular can act as a unique ‘biomarker signature’ and help identifying releasing cells and their circumstances.

While knowledge of the extensive target repertoire of individual miRNAs greatly expanded our understanding of miRNA functions, the high resolution down to single nucleotide alterations enabled by small-RNA sequencing technologies added an entire new layer of complexity by detecting a great abundance of miRNA isoforms. These isomiRs show significant sequence and length heterogeneity, generated by base exchanges from the canonical sequence, and/or additions and/or deletions at the 3’ or 5’ end. The quantified isomiRs can change the mRNA targeting behaviour, either subtly through supplementary or complementary binding at the 3’ end, or drastically by altering the seed sequence at the 5’ end. By taking these target gene shifts into account and using a consensus of predicted genes that reflects actual miRNA sequence distribution and binding efficiencies, the accuracy of predicted pathways and related diseases is greatly improved. To realize this approach we implement these ideas in a ‘bioinformatical pipeline’ which analyses the small RNA sequencing read data files in a fully automatic way.

The assessment of mRNA targets can help discern the physiological relevance of identified miRNA biomarkers and even uncover novel relationships. We apply this ‘analysis pipeline’ to discover new integrative mRNA/miRNA regulation pattern in agri-veterinary research and to predict new ‘miR biomarker signatures’ in human clinical diagnostics.

Epitranscriptomic regulation of non-coding RNAs

Stefan Ludwig Ameres

IMBA, Austria

For RNA to fulfil its essential function within the cellular environment, numerous chemical modifications have evolved to sculpt its physical and functional interactions. Although more than hundred types of RNA modifications have built the descriptive foundation of what is referred to as the epitranscriptome, their mode of action remains largely unknown. I will present our results on the function of chemical RNA modifications at the intersection of small RNA silencing pathways and general RNA metabolism.

Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. We identified the first RNA-specific uridylytransferase that is required for the majority of 3ʹ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3ʹ overhang of microRNA hairpins, which regulates processing by Dicer and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3ʹ guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3ʹ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3ʹ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila. Our data also provide an atlas of post-transcriptional modifications in small RNAs and their precursors in flies, providing a framework for understanding the epitranscriptomic regulation of small RNA biogenesis and function.

We could also show that uridylation in flies triggers the processive 3ʹ-to-5ʹ exoribonucleolytic decay via the ribonuclease II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms a stable cytoplasmic uridylation-triggered RNA processing (TRUMP) complex, that functionally cooperates in the degradation of structured RNAs in vitro, providing a molecular explanation for the inhibition of mirtron maturation in flies. RNA-immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including long non-coding RNA, such as rRNA, the essential RNase MRP and the signal recognition particle RNA 7SL. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R our results imply a conserved molecular function of RNase II/R enzymes as ‘readers’ of destabilizing post-transcriptional marks – uridylation in eukaryotes and adenylation in prokaryotes – that play important roles in non-coding RNA surveillance.

Extracellular Vesicles and Their Associated Cargos in Health and Disease.

Mirco Castoldi

University of Düsseldorf, Germany

One of the major challenges towards an improved treatment of human diseases is the identification of appropriate prognostic and diagnostic markers. During the past decade, microRNA (miRNA) activity has been associated with the control of a wide range of cellular processes. Importantly, it has been shown that dysfunctional expression of specific miRNAs is associated with the development of a variety of diseases in human. However, the inherent difficulties associated to the collection of diseased material from patients has severely limited the investigation of cellular miRNAs as source for disease-related biomarker. Remarkably, the discovery of populations of cell-free miRNAs circulating in the blood of healthy as well as diseased individuals, have raised the possibility to identify specific signature reflecting clinical manifestation of diseases. Although two different sub-populations of circulating miRNAs exist, we will specifically focus on the clinical utility, isolation, and detection of extracellular vesicles-associated miRNAs. Extracellular vesicles (EVs) are a heterogeneous group of nano-sized particles, which play a key role in inter-cellular communication and signaling. EVs are secreted by most, if not all, cell types composing the organism. Compelling evidences indicate that both the quantitative and qualitative composition of EVs may correlate with the diseased state of the patient. However, application of experimental protocols to the clinical setting is a challenge, requiring the establishment of easy to use, affordable and robust methodologies. Here, implementation of experimental pipelines required for the isolation and analysis of EVs and EVs-associated miRNAs from patients’ serum in the context of diagnostic laboratories will be presented and discussed.

In silico functional analysis of microRNA : Towards the characterisation of miRNAs as biomarkers.

Artemis Hatzigeorgiou

Univeristy of Thessaly, Greece

RNA revolution has turned non-coding RNAs (ncRNAs) from dark-matter into a biological research hotspot. ncRNA families, such as microRNAs (miRNAs) and more recently, long non coding RNAs (lncRNAs) are being researched for physiological and pathological implications. The role of miRNAs in development and diseasehas been widely reported during the past few years.

During the talk tools from the DIANA suite of miRNA analysis (microrna.gr) will be presented that can be used as basic step for the identification of miRNA biomarkers.

Such tools and databases include miRNA target prediction (DIANA micro-T) and annotation (TarBase, LNCBase), pathway analysis (DIANA–mirPath), regulation of microRNAs (miRGEN) and the use of RNAseq data for the identification of key regulatory components (miRNA and/or Transcription factors) based on two investigated stages (DIANA- mirExTra). The DIANA suite is widely used with more than 100,000 unique users per year from all over the world.

A novel assay system for improved specificity and sensitivity of miRNA detection for all the major model organisms

Simon Baker

Bioline Reagents Limited, United Kingdom

For some time now, Bioline has offered an extensive list of individual human miRNAs, using the proprietary algorithm developed by MiRXES to maximize miRNA detection sensitivity, while minimizing non-specific interactions. The versatility of the assay design system has now allowed us to extend the range to include all the 27,000 miRNA listed on miRBase, providing plant and animal miRNA assays. The resulting real-time PCR assays enable detection of extremely low levels of miRNA with high specificity using a SYBR® Green detection chemistry, allowing the discrimination between closely related miRNA sequences. All EPIK miRNA Select Assays have been validated using synthetic miRNA templates and human assays have also been validated against total RNA. Typically the assays detect as few as 100 copies of template per RT reaction with excellent assay efficiency and linearity. We will also discuss the appropriate uses of positive controls to normalize results between assays and between experiments, and how the EPIK RNA Spike-In controls can be used to build an accurate picture of the relative concentration of miRNA in a sample. These controls can be particularly effective in addressing complex biological problems such as the abundance of miRNA in exosomes from human blood.

Two-tailed RT-qPCR: a Novel Method for Highly Accurate miRNA Quantification

Lukas Valihrach1, Peter Androvic1, Julie Elling2, Robert Sjoback2, Mikael Kubista1,2

1Institute of Biotechnology AS CR, Czech Republic; 2TATAA Biocenter AB, Sweden

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. MiRNAs are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, 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 8 logs and a sensitivity sufficient to detect down to a hundred of target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (R2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

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