Using FirePlex™ Particle Technology for Multiplex MicroRNA Profiling Without RNA Purification

Abstract

Accuracy of miRNA profiling is enhanced when sample processing can be kept to a minimum, avoiding steps such as RNA purification that can introduce bias and inaccuracies. Here we describe a novel multiplex circulating miRNA assay that enables the profiling of up to 65 miRNAs of choice in the same well directly from plasma (including heparin plasma) or serum, with no need for RNA purification. The main component of the assay is FirePlex™ hydrogel particles, which enable the multiplex capture of miRNAs with picomolar sensitivity and high specificity. Results are obtained using conventional flow cytometry and easy to use software, which allows fast analysis and interpretation of the experimental data. This chapter provides methods to profile miRNAs with PCR sensitivity from as little as 10 μL of crude biofluid sample, or from less than 100 pg of purified RNA.

Keywords: High-throughput; Hybridization; Multiplex; Polymerase chain reaction; microRNA.