MicroRNAs are short (18-23 nucleotides) noncoding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA-expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter, we present a fluorescence-based detection method that allows the combination of microRNA in situ hybridization with immunohistochemical staining of one and, in this updated version of the paper, two protein markers detected with primary antibodies raised in the same host species.
Keywords: Fluorescence multiplexing; Immunohistochemistry; In situ hybridization; Locked nucleic acid; MicroRNA.