用于研究TDP43剪接功能的高通量流式细胞术检测
High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function.
作者信息
Schmidt H Broder, Rohatgi Rajat
机构信息
Department of Biochemistry, Stanford University School of Medicine, Stanford, USA.
Department of Medicine, Stanford University School of Medicine, Stanford, USA.
出版信息
Bio Protoc. 2020 Apr 20;10(8):e3594. doi: 10.21769/BioProtoc.3594.
Abstract
Mutations in RNA-binding proteins (RBPs) such as TDP43 are associated with transcriptome-wide splicing defects and cause severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The impact of RBP mutations on splicing function is routinely studied using PCR-based bulk measurements. However, the qualitative and low-throughput nature of this assay make quantitative and systematic analyses, as well as screening approaches, difficult to implement. To overcome this hurdle, we have developed a quantitative, high-throughput flow cytometry assay to investigate TDP43 splicing function on a single-cell level.
摘要
诸如TDP43等RNA结合蛋白（RBP）的突变与全转录组范围的剪接缺陷相关，并导致严重的神经退行性疾病，包括肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）。RBP突变对剪接功能的影响通常使用基于PCR的大量测量方法进行研究。然而，该检测方法的定性和低通量特性使得定量和系统分析以及筛选方法难以实施。为了克服这一障碍，我们开发了一种定量、高通量的流式细胞术检测方法，以在单细胞水平上研究TDP43的剪接功能。
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