In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets

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• 作者：Gerard J. Nuovo
• 关键词：MicroRNA precursor ; In situ hybridization ; In situ PCR ; LNA probes ; Immunohistochemistry
• 刊名：Methods
• 出版年：2010
• 出版时间：December 2010
• 年：2010
• 卷：52
• 期：4
• 页码：307-315
• 全文大小：2154 K

文摘

This manuscript details a protocol for the co-localization of a microRNA and its putative protein target in paraffin embedded formalin fixed tissues. The key variables for the first step, microRNA in situ hybridization, includes probe concentration (1–2 pmol/μl), locked nucleic acid (LNA) modified probes, protease digestion (pepsin 1.3 mg/ml), and a low stringency wash. Key variables for the subsequent immunohistochemical step are the concentration of the primary antibody, proper pretreatment (none, proteinase K, or antigen retrieval), and use of a highly sensitive detection system. A computer based system can convert the colorimetric signals (blue chromogen (NBT/BCIP) for the microRNA, and either a red (fast red) or brown (DAB) chromogen for the protein) to distinct fluorescent-based colors, and then mix them to determine if a given cell has the microRNA and protein of interest. Co-expression of a microRNA and its putative target in tissue sections offers physiologic corroboration of solution-based methods that a given microRNA may be regulating a specific protein.