MicroRNA in situ hybridization.

In situ hybridization (ISH) is a technology that allows detection of specific nucleic acid sequences in tissue samples at the cellular level. For detection of individual microRNAs (miRNAs) and mRNAs, the ISH technology determines the cellular origin of expression and provides information on expression levels in different tissue compartments and cell populations. This histological expression analysis is of crucial importance for elucidating roles particularly of miRNAs in molecular and biological processes. mRNA expression analyses can partly be replaced by immunohistochemical detection of the protein encoded by the mRNA. Combined with the short sequences of the miRNAs (18-22 bp), this leaves miRNA ISH as an indispensable yet challenging technology in terms of detection and specificity analysis. In this chapter, a simple miRNA ISH protocol using chromogenic detection is presented. I touch upon critical steps in the ISH protocol, different applications on ISH technology platforms, advantageous use of locked nucleic acids (LNA™) in miRNA detection probes, qualification of clinical paraffin samples, and specificity analyses and quantification of the ISH signal.