Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization.
Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization.
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DOI：
10.3791/57920
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发表时间：
2018-09
期刊：
影响因子：
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通讯作者：
F. Memi;D. Tirziu;I. Papangeli
中科院分区：
文献类型：
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作者：
F. Memi;D. Tirziu;I. Papangeli
micro-RNA（miRNAs）是单链RNA转录物，其与信使RNA（mRNA）结合并抑制其翻译或促进其降解。迄今为止，miRNA已参与了大量的生物学和疾病过程，这意味着需要可靠的检测miRNA转录本的方法。在这里，我们描述了一个详细的协议地高辛标记（DIG）锁核酸（LNA）探针为基础的miRNA检测，结合蛋白质免疫染色小鼠心脏切片。首先，我们使用探针进行原位杂交技术，以确定miRNA-182在对照组和心肌肥厚小鼠心脏切片中的表达。接下来，我们对心肌肌钙蛋白T（cTnT）蛋白进行免疫染色，在相同的切片上，以使miRNA-182与心肌细胞共定位。使用该方案，我们能够通过基于碱性磷酸酶的比色测定法检测miRNA-182，并通过荧光染色检测cTnT。该方案可用于通过DIG标记的LNA探针检测任何感兴趣的miRNA的表达，以及小鼠心脏组织切片上的相关蛋白表达。
micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.