MicroRNA Profiling Using a Rapid and Highly Sensitive qPCR Panel

Recent studies have shown that micro RNA molecules (miRNA) are released from tissues and cells into circulation where their expression correlates with specific disease and non-disease conditions. Analysis of miRNA is attractive as these molecules are stabilized by protein-binding or vesicle encapsulation (such as exosomes) and provide access to humoral state without the need for sequencing the entire human transcriptome. These attributes have made circulating miRNAs promising non-invasive candidate biomarkers for the early detection of disease, prognosis or treatment selection. However, significant technological challenges remain. These include data inconsistencies across studies which has hampered the development of accurate and robust miRNA-based tests. Efficient identification of miRNA biomarkers requires a well-designed study and an integrated workflow with consistent sample extraction, robust RT-qPCR quantification and stringent statistical analysis. In addition, control measures are essential to monitor and normalize technical or biological variations that may obstruct the interpretation of data. Bioline developed an enabling platform based on a proprietary design concept and validated the resulting qPCR assays against both clinical samples and synthetic RNA constructs. They detected extremely low levels of circulating miRNA with high sensitivity and specificity. This new platform has been used for the routine detection of over 400 miRNAs, using low input material (e.g. 200 microliters of serum and plasma) with a dynamic range span over eight orders of magnitude. The total workflow variation was less than 0.5 Cq. The platform has been validated independently by molecular diagnostic and pharmaceutical laboratories and showed superior performance to existing commercial assays. This highly reproducible, sensitive platform has been developed to produce complete systems for miRNA profiling of cancer samples, biofluids and stem cells.

Effective miRNA profiling calls for reproducible, sensitive and specific tools with turn-around times fast enough to support investigations into what can be a rapidly changing disease progression and treatment environment. Bioline introduced the latest miRNA RT-qPCR technology, including the EPIK™ Cancer miRNA Panel, offering sensitive SYBR® Green-based detection for 352 targets, including the most differentially expressed miRNAs and controls, for convenient, robust and extremely specific miRNA analysis. As efforts continue towards the transition of miRNA profiling from bench to bedside, RT-qPCR panels offer an ideal tool to support current applied research and allowing access to the underpinning clinical processes. The latest technology in the field is represented by the strong assay design of the EPIK™ Cancer miRNA Panel, covering the most useful targets to monitor disease states and offering both the performance and the sensitivity required for low level starting materials or liquid biopsy applications.

Speaker

Simon Baker PhD, Global R&D Director, Bioline Reagents Ltd

Simon Baker gained his Ph.D. in Biological Sciences from the University of Warwick in 1991, and then focused on molecular biology and biochemistry via a Royal Society European Fellowship at the University of Groningen in the Netherlands. He returned to the UK to the University of Oxford, where he worked in the Department of Biochemistry with Professor Stuart Ferguson and then in the Department of Engineering with Professor Chris Knowles. It was here he began consultancy in the biotechnology industry working with what was then a small biotech company in Epsom called ABgene. He has since worked with Thermo Fisher, TMO Group, The Neal Partnership, 4titude and many others. After several years as a Lecturer in Microbiology at Birkbeck, University of London, and then Senior Lecturer in Biotechnology at Oxford Brookes University, he joined Bioline as full time Director of R&D in June 2012. He is now Senior Director of Bioline’s R&D groups throughout the world, guiding the company’s developments in PCR-related technologies, sample preparation, micro RNA detection and next generation sequencing.

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