Development and Optimisation of PCR Assays to Analyse MicroRNAs and their Target Genes

David Arthur Simpson Queen’s University Belfast, United Kingdom

Abstract
Introduction: Endothelial progenitor cells (EPCs) isolated from blood release microRNA-containing extracellular vesicles (EVs) which can potentially be harnessed to modulate angiogenesis as a treatment for vascular disease. These EVs contribute to the pool of circulating microRNAs, which provide biomarkers for many conditions. Quantification of microRNAs is required both to study their role in vascular repair and to exploit their potential as biomarkers. Multiple issues need to be considered in the design of a successful assay. RT-PCR reagents and template concentrations must be optimised. The final reaction volume must be minimised to reduce reagent use and amount of template required. Increasing the rate of thermal cycling brings time-savings and can be critical for certain clinical applications. The specificity of the assay with regard to microRNA isomiRs must be defined.
Methods: Probe-based assays and polyadenylation followed by oligo-dT primed reverse transcription and PCR with SYBR Green were employed. PCR reactions were set up using an Echo liquid handler (Labcyte), which uses acoustic energy to transfer 25nl droplets. qPCR was performed using a 384 well LightCycler 480 (Roche) or a rapid thermal cycler (xxpress, BJS Biotechnologies). RT-PCR products were sequenced using the Ion Torrent platform (Life Technologies).
Results: The quality of data was maintained as qPCR volumes were reduced to 2 µl. MicroRNAs can be amplified from plasma in less than 10 min using the xxpress cycler. Sequencing of RT-PCR products provides a profile of isomiRs for specific microRNAs comparable to sequencing of entire small RNA libraries.
Conclusion: The ability to accurately transfer nanolitre volumes and therefore adopt very low reaction volumes facilitates rapid optimisation of PCR reaction conditions and saves reagents and template. Characterisation of microRNA isomiRs and rapid detection of them from plasma broadens their potential as clinical biomarkers.