MicroRNA In Situ Hybridization in Paraffin-Embedded Cultured Cells

MicroRNA-21 (miR-21) is one of the most abundant microRNAs in cancer tissues and is considered a strong prognostic biomarker. In situ hybridization (ISH) analyses using locked nucleic acid (LNA) probes have shown that miR-21 is expressed in stromal fibroblastic cells and in subsets of cancer cells. Image analysis of the miR-21 ISH signal has shown that increased expression estimate is associated with poor prognosis in colon cancer. However, assessment of the ISH signal by image analysis to obtain quantitative estimates has been done in retrospective studies without normalization of the expression estimates to reference parameters. The ISH signal output is sensitive to several experimental parameters, including hybridization temperature, probe concentration, and pretreatment, and therefore improved standardized procedures are warranted. We considered the use of paraffin-embedded cultured cells (PECCs) as reference standards that potentially can accompany staining of clinical cancer samples. We found that the cancer cell lines HT-29, CACO-2, and HeLa cells express miR-21 when measured by ISH, and used those cell lines to obtain PECCs. In this methods chapter we present a fixation and embedding procedure to obtain PECCs suitable for microRNA ISH and a double-fluorescence protocol to stain microRNAs together with protein markers in the PECCs.