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Workpackage 5: Assay development: miRNA

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Objectives
  • To show feasibility of multiplexed miRNA testing on a novel multiplex biomarker analysis platform (Evalution™ by Biocartis) with relevant analytical performance
  • To design and develop the final miRNA multiplex assay and transfer the Biocartis dedicated kits to the University Clinic Hamburg-Eppendorf Laboratory (Partner 1) for testing of clinical samples
Description of Work

The miRNA assay will be developed to provide the absolute concentration of multiple relevant miRNAs in serum samples and therefore to assess their value as biomarkers for the prediction of cardiovascular risk.
During the initial phase of this WP, the assay performance requirements and the assay format are defined. The miRNAs of interest, extracted from serum, will be reverse-transcribed, amplified and subsequently detected in Evalution™ in a quantitative way, with high specificity and broad dynamic range. To show feasibility of the approach, a 3-plex assay will be designed and the analytical performances characterized with synthetic miRNAs. The full development of the assay will start upon communication of the final miRNA panel, when established by UKE and approved by the consortium. Most of the optimization phase will be devoted to the design of the multiplex amplification and the definition of the controls. The RT-PCR is based on the combined action of miRNA-specific and universal primers in a single tube. The overall protocol ensures that quantitative relations are maintained among different miRNAs throughout the reaction. The amplified material will be analyzed in Biocartis' multiplex analysis platform, which enables fast real-time detection of multiple analytes in a single microfluidic channel. Highly specific self-reporting probes with catalytic activity will be designed for each individual miRNA and coupled to digitally encoded microparticles. The detection of target miRNAs is based on the fluorescence signal produced upon recognition events. The concentration of each miRNA can be extrapolated from the real time detection curves, as a known mathematical relation correlates the kinetics of detection with the target concentration. Dedicated software will be developed to provide the concentration of each targeted miRNA, corrected for the yield of extraction.
Upon completion of the optimization phase, the assay will be transferred to Partner 1, together with the instrument and related consumables, to validate the panel of miRNA biomarkers in a case cohort of 20000 samples.

Partners involved

Biocartis Lausanne (WP coordinator), UKE Hamburg