前列腺癌临床组织中的微小RNA表达分析
miRNA Expression Analyses in Prostate Cancer Clinical Tissues.
作者信息
Bucay Nathan, Shahryari Varahram, Majid Shahana, Yamamura Soichiro, Mitsui Yozo, Tabatabai Z Laura, Greene Kirsten, Deng Guoren, Dahiya Rajvir, Tanaka Yuichiro, Saini Sharanjot
机构信息
Department of Urology, Veterans Affairs Medical Center, San Francisco, University of California San Francisco.
Department of Urology, Veterans Affairs Medical Center, San Francisco, University of California San Francisco;
出版信息
J Vis Exp. 2015 Sep 8(103):53123. doi: 10.3791/53123.
A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).
前列腺癌（PCa）临床管理中的一个关键挑战是目前用于疾病筛查、诊断、预后和治疗的生物标志物存在不足。近年来，微小RNA（miRNA）已成为前列腺癌诊断和预后有前景的替代生物标志物。然而，将miRNA开发为前列腺癌的有效生物标志物在很大程度上依赖于在临床组织中对其进行准确检测。由于肿瘤异质性、采样误差、基质污染等原因，对前列腺癌临床标本进行miRNA分析往往具有挑战性。本文的目的是描述一种简化的工作流程，用于使用定量实时PCR（RT-PCR）和原位杂交（ISH）相结合的方法，对存档的福尔马林固定石蜡包埋（FFPE）或新鲜冷冻的前列腺癌临床标本进行miRNA分析。在这个工作流程中，我们优化了从FFPE和冷冻前列腺组织中提取miRNA以及通过基于Taqman探针的miRNA RT-PCR进行表达分析的现有方法。此外，我们描述了一种使用基于锁核酸（LNA）的探针在前列腺组织中进行ISH分析以检测miRNA的优化方法。我们优化的miRNA ISH方案可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA）。
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