Analysis of microRNA niches: techniques to measure extracellular microRNA and intracellular microRNA in situ.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that negatively regulate gene expression and control a wide range of cellular processes. Extracellular forms of miRNA circulating in the bloodstream (circulating miRNA, c-miRNA) are of increasing interest for their potential as biomarkers and long-range physiological signaling molecules. Precise measurement of intracellular miRNA expression is possible but can be challenging, especially in the context of specialized tissue niches in vivo. The accurate measurement of extracellular miRNA presents other obstacles stemming from their low concentrations and confounding sources of intracellular miRNA that contaminate RNA extraction protocols. Here, we describe multiple methods to isolate extracellular miRNA from cell culture media, serum, and plasma in order to accurately measure their variable expression under different conditions. We additionally describe an in situ staining protocol designed to not only quantify but also localize miRNA in formalin-fixed paraffin-embedded tissue that may prove useful in describing the action of c-miRNA before they leave their tissue of origin and after they potentially arrive at their target destination.