RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
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DOI：
10.1261/rna.070177.118
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发表时间：
2019-09-01
期刊：
影响因子：
4.5
通讯作者：
Harper, Scott Q.
中科院分区：
文献类型：
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作者：
Chermahini, Gholamhossein Amini;Rashnonejad, Afrooz;Harper, Scott Q.
面肩肱型肌营养不良症 (FSHD) 是最常见的肌营养不良症之一。 FSHD 是由肌肉中有毒 DUX4 基因的异常表达引起的。由于 DUX4 表达水平较低，使用传统方法检测患者组织中的内源性 DUX4 可能具有挑战性。因此，开发简单可靠的DUX4检测方法是FSHD领域的重要需求。在这里，我们描述了这样一种方法，它使用 RNAscope 测定，一种 RNA 原位杂交 (ISH) 技术。我们证明，定制设计的 RNAscope 测定可以检测转染的 HEK293 细胞中过表达的 DUX4 mRNA 以及 FSHD 患者来源的肌管中的内源性 DUX4 mRNA。 RNAscope 测定对于跟踪使用我们的治疗性 mi405 microRNA 治疗后 DUX4 mRNA 的减少高度敏感，这表明基于 RNAscope 的 DUX4 表达测定可以开发为治疗试验中的前瞻性结果测量。这项研究可以为优化和开发一种新的、基于 RNA ISH 的快速分子诊断检测方法奠定基础，以供未来 FSHD 领域的临床使用。
Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. Therefore, developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed DUX4 mRNA in transfected HEK293 cells and endogenous DUX4 mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in DUX4 mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based DUX4 expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field.