Abstract
Abstract To address the needs for circulating miRNA biomarker validation, we developed the Multiplexed Circulating microRNA assay. This assay enables the detection of up to 68 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. The Circulating microRNA assay combines particle¬-based multiplexing, using patented Firefly hydrogel particles, with single¬ step RT-PCR signal amplification using universal primers. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target¬-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating 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 validation 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 Firefly Circulating microRNA Assay Fixed Panels. Together, this novel combination of bioinformatics tools and multiplexed, high¬-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens. Citation Format: Isaac Stoner, Michael Tackett, Conor Rafferty, Andreas Windemuth, Elnaz Atabakhsh, Jessica Tytell, Daniel Pregibon. High-throughput, purification-free, multiplexed profiling of circulating miRNA for discovery, validation, and diagnostics. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B30.
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