Quantification of Tissue and Circulating MicroRNAs by Droplet Digital PCR

MicroRNAs (miRNAs) are gene expression regulators that play a fundamental role in developmental and biological processes. Dysregulated expression of miRNAs has been associated with most human conditions, and their accurate quantification is an essential activity in molecular biology laboratories. Indeed, the levels of specific tissue or circulating miRNAs have been tested as disease biomarkers in many clinical and experimental settings. Droplet digital PCR (ddPCR) technology is a sensitive and accurate method to obtain the absolute or relative quantification of specific miRNAs, bypassing several issues related to low abundance targets and PCR efficiency. This chapter addresses the workflow and methods for miRNA quantification in formalin-fixed paraffin-embedded (FFPE) samples, cells, and plasma/serum/extracellular vesicles using EvaGreen-based ddPCR or probe-based ddPCR, as well as how to analyze and interpret results. In addition, we provide a ddPCR method to quantify miRNA isoforms (isomiRs).