Sempere Lorenzo F
Program in Skeletal Disease and Tumor Microenvironment, Laboratory of microRNA Diagnostics and Therapeutics, Center for Cancer and Cell Biology, Van Andel Institute, 333 Bostwick Ave., N.E., Grand Rapids, MI, 49503, USA,
Methods Mol Biol. 2014;1211:151-70. doi: 10.1007/978-1-4939-1459-3_13.
The application of locked nucleic acid chemistry for microRNA detection by in situ hybridization, and thereby visualization of microRNA expression at single-cell resolution, has contributed to our understanding of the roles that these short noncoding regulatory RNAs play during development, physiology, and disease. Several groups have implemented chromogenic-based and fluorescence-based protocols to detect microRNA expression in formalin-fixed paraffin-embedded clinical tissue specimens. These emerging robust and reproducible tissue slide-based assays are valid tools to bring about the clinical application of in situ microRNA detection for routine diagnostics. Here, I describe a fully automated fluorescence-based four-color multiplex assay for co-detection of a microRNA (e.g., let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210), reference RNA (e.g., U6 snRNA, 18S rRNA), and protein markers (e.g., CD11b, CD20, CD45, collagen I, cytokeratin 7, cytokeratin 19, smooth muscle actin, tubulin, vimentin) in FDA-approved Leica Bond-MAX staining station.
将锁核酸化学应用于原位杂交检测微小RNA，从而在单细胞分辨率下可视化微小RNA的表达，有助于我们理解这些短链非编码调节性RNA在发育、生理学和疾病过程中所起的作用。多个研究小组已经实施了基于显色和基于荧光的方案，以检测福尔马林固定石蜡包埋临床组织标本中的微小RNA表达。这些新兴的、强大且可重复的基于组织切片的检测方法，是实现原位微小RNA检测在常规诊断中临床应用的有效工具。在此，我描述一种基于荧光的全自动四色多重检测方法，用于在经美国食品药品监督管理局（FDA）批准的徕卡Bond-MAX染色工作站中共同检测一种微小RNA（例如，let-7a、miR-10b、miR-21、miR-34a、miR-126、miR-145、miR-155、miR-205、miR-210）、参考RNA（例如，U6小核RNA、18S核糖体RNA）和蛋白质标志物（例如，CD11b、CD20、CD45、胶原蛋白I、细胞角蛋白7、细胞角蛋白19、平滑肌肌动蛋白、微管蛋白、波形蛋白）。
