NTTS 2017

To address the need for circulating miRNA biomarker discovery and verification, we developed the FirePlex^® microRNA assay. This assay enables multiplex detection of up to 65 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. This assay combines particle­-based multiplexing, using patented FirePlex^® hydrogel particles, with single­ step RT-PCR signal amplification using universal primers. Thus, the FirePlex microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target­-specific qPCR. The assay can reliably detect as few as 1000 microRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification, making the assay ideally suited for profiling in serum, plasma or urine. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the FirePlex microRNA assay are displayed and interpreted using our included Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid discovery and verification of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating and tumor microRNA profiles using the FirePlex microRNA Assay. Together, this novel combination of bioinformatics tools and multiplexed, high­-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens.