Fully automated fluorescence-based four-color multiplex assay for co-detection of microRNA and protein biomarkers in clinical tissue specimens.
Fully automated fluorescence-based four-color multiplex assay for co-detection of microRNA and protein biomarkers in clinical tissue specimens.
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DOI：
10.1007/978-1-4939-1459-3_13
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发表时间：
2014
影响因子：
--
通讯作者：
L. Sempere
中科院分区：
文献类型：
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作者：
L. Sempere
应用锁核酸化学原位杂交检测microRNA，从而可视化microRNA表达在单细胞分辨率，有助于我们了解这些短的非编码调控RNA在发育，生理和疾病过程中发挥的作用。几个研究小组已经实施了基于显色和基于荧光的方案来检测福尔马林固定石蜡包埋的临床组织标本中的microRNA表达。这些新兴的稳健且可重复的基于组织载玻片的测定是将原位microRNA检测用于常规诊断的临床应用的有效工具。在这里，我描述了一种全自动的基于荧光的四色多重测定法，用于共同检测微小RNA（例如，let-7a，miR-10 b，miR-21，miR-34 a，miR-126，miR-145，miR-155，miR-205，miR-210），参考RNA（例如，U6 snRNA、18 S rRNA）和蛋白质标记物（例如，CD 11b、CD 20、CD 45、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）。
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.