微小RNA的杂交（原位杂交）与量子点
Hybridization (ISH) and Quantum Dots (QD) of miRNAs.
作者信息
Josson Sajni, Gururajan Murali, W K Chung Leland
机构信息
Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA.
Present address: Neostrata Inc, Princeton, USA.
出版信息
Bio Protoc. 2017 Feb 20;7(4):e2138. doi: 10.21769/BioProtoc.2138.
miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.
微小RNA（miRNA）是短链非编码RNA，可抑制信使核糖核酸（mRNA）的翻译。miRNA调节多种细胞过程。已知某些miRNA可诱导肿瘤发生。miRNA可通过实时聚合酶链反应（PCR）进行检测，并可使用杂交（原位杂交，ISH）和量子点（QD）的组合进行成像。使用量子点的优势在于可通过多重量子点同时检测多种miRNA。此外，miRNA可在组织的不同区域可视化。由于miRNA是各种疾病状态的生物标志物，因此可在组织切片中对其进行可视化和定量分析，以用于诊断和预后评估。在此，我们描述组织切片的ISH-QD分析。使用来自异种移植物或临床标本的组织切片。将这些切片脱石蜡，用蛋白酶K处理，并用针对miRNA的生物素探针进行杂交。杂交通过标记生物素探针进行，随后用链霉亲和素标记的量子点进行标记。对量子点进行图像采集，并分析miRNA的表达水平。将ISH和QD相结合，为检测组织中不同细胞中的miRNA提供了一种强大的工具。
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