Assessment of tissue distribution and subcellular localization of miR-302 and miR-21 by means of in situ hybridization technique

Abstract

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellularrnprocesses, and their disregulations have been linked to several pathologic conditions, mainly cancers.rnDetermining tissue distribution of miRNAs is a prerequisite for understanding their exact functions duringrndevelopment, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate thernsub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the importantrnrole of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAsrn(miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotentrnembryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks,rnproteinase K digestion, probe concentration, antibody development and light sensitive color reaction werernoptimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detectedrnin the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples ofrnseminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detectionrnin FFPE samples and NT2 cell line.