Cerebrospinal fluid MicroRNA profiling using quantitative real time PCR.
Cerebrospinal fluid MicroRNA profiling using quantitative real time PCR.
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DOI：
10.3791/51172
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发表时间：
2014-01
期刊：
影响因子：
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通讯作者：
Marco Pacifici;S. Delbue;Ferdous Kadri;F. Peruzzi
中科院分区：
文献类型：
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作者：
Marco Pacifici;S. Delbue;Ferdous Kadri;F. Peruzzi
MicroRNA (miRNA) 通过引导 RISC 到达 mRNA 上的靶位点，从而调节其翻译抑制，从而构成有效的基因调控层。 miRNA 表达的变化已被证明与所有主要复杂疾病的发展有关。此外，最近的研究结果表明，miRNA可以分泌到细胞外环境并进入血液和其他体液，在那里它们可以高度稳定地循环。此类循环 miRNA 的功能在很大程度上仍然难以捉摸，但系统性高通量方法（例如 miRNA 分析阵列）已导致在多种病理条件下识别 miRNA 特征，包括神经退行性疾病和几种类型的癌症。在这种情况下，正如我们最近的研究所报告的那样，脑脊液中 miRNA 表达谱的鉴定使 miRNA 成为生物标志物分析的有吸引力的候选者。有多种工具可用于分析 microRNA，例如微阵列、定量实时 PCR (qPCR) 和深度测序。在这里，我们描述了一种通过定量实时 PCR 分析脑脊液中 microRNA 的敏感方法。我们使用了 Exiqon microRNA 即用型 PCR 人类面板 I 和 II V2.R，它可以检测 742 种独特的人类 microRNA。我们对阵列进行三次重复运行，并使用 GenEx Professional 5 软件处理和分析数据。使用该协议，我们成功地分析了各种类型的细胞系和原代细胞、脑脊液、血浆和福尔马林固定石蜡包埋组织中的 microRNA。
MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis. There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software. Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.