In Situ Detection of MicroRNAs in Paraffin-Embedded, Formalin-Fixed Tissues: Different Methodologies and Co-localization with Possible Targets

The in situ detection of microRNAs (miRNAs) has been greatly facilitated by several recent technologic advances. These include the locked nucleic acid (LNA)-modified nucleotide, in situ extension of the mature miRNA, and RT in situ PCR amplification of the pre- and pri-microRNA. Furthermore, marked increases in the sensitivity of automated immunohistochemistry allow for co-localization of a given miRNA and its possible target. Key variables for successful LNA in situ detection of miRNAs include probe concentration, miRNA copy number, and stringency of the hybridization and wash. Success with RT in situ PCR and ultramer extension is highly dependent on adequate protease digestion. The key variables with immunohistochemistry include antibody concentration and pretreatment conditions. With co-labeling, miRNA detection is done first; the antigen can be co-localized if the immunohistochemistry reaction requires pretreatment in either the protease or antigen retrieval.