Analysis of microRNA niches: techniques to measure extracellular microRNA and intracellular microRNA in situ.
Analysis of microRNA niches: techniques to measure extracellular microRNA and intracellular microRNA in situ.
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DOI：
10.1007/978-1-62703-453-1_12
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Chan, Stephen Y
中科院分区：
文献类型：
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作者：
Parikh, Victoria N;Chan, Stephen Y
MicroRNA（miRNA）是一种非编码的小RNA分子，负调控基因表达并控制广泛的细胞过程。在血流中循环的细胞外形式的miRNA（循环miRNA，c-miRNA）由于其作为生物标志物和长距离生理信号分子的潜力而越来越受到关注。细胞内miRNA表达的精确测量是可能的，但可能具有挑战性，特别是在体内专门组织小生境的背景下。细胞外miRNA的准确测量存在其他障碍，这些障碍源于它们的低浓度和污染RNA提取方案的细胞内miRNA的混杂来源。在这里，我们描述了从细胞培养基、血清和血浆中分离细胞外miRNA的多种方法，以准确测量它们在不同条件下的可变表达。我们还描述了一种原位染色方案，不仅用于定量，而且用于在福尔马林固定石蜡包埋（FFPE）组织中定位miRNA，这可能有助于描述c-miRNA在离开其组织来源之前和可能到达其靶目的地之后的作用。
MicroRNA (miRNA) are small, non-coding RNA molecules that negatively regulate gene expression and control a wide range of cellular processes. Extracellular forms of miRNA circulating in the bloodstream (circulating miRNA, c-miRNA) are of increasing interest for their potential as biomarkers and long-range physiological signaling molecules. Precise measurement of intracellular miRNA expression is possible but can be challenging, especially in the context of specialized tissue niches in vivo. The accurate measurement of extracellular miRNA presents other obstacles stemming from their low concentrations and confounding sources of intracellular miRNA that contaminate RNA extraction protocols. Here, we describe multiple methods to isolate extracellular miRNA from cell culture media, serum, and plasma in order to accurately measure their variable expression under different conditions. We additionally describe an in situ staining protocol designed not only to quantify, but also to localize miRNA in formalin-fixed paraffin-embedded (FFPE) tissue, that may prove useful in describing the action of c-miRNA before they leave their tissue of origin and after they potentially arrive at their target destination.