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Updated: Jun 30, 2025

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Published on: August 26, 2018

高通量RNA-HCR-FISH检测内源性预mRNA拼接变体的检测

Asaf Shilo¹, Gianluca Pegoraro², Tom Misteli³

¹Cell Biology of Genomes, Center for Cancer Research (CCR), National Cancer Institute, NIH, Bethesda, MD, USA.

Methods in molecular biology (Clifton, N.J.)

|March 19, 2024

概括

HiFENS (内源性前mRNA拼接异型的高通量FISH检测) 可视化单细胞中的mRNA拼接变体. 这种方法增强了RNA-FISH灵敏度,改善了基因表达和拼接途径的发现.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
细胞生物学细胞生物学

背景情况:

在细胞中可视化RNA的过程中,RNA-光在位杂交 (RNA-FISH) 非常重要.
由于信号限制,现有的RNA-FISH方法在检测低丰度的mRNA拼接变体方面面临挑战.
功能性基因组学需要对内源性mRNA前拼接异型进行高通量检测.

研究的目的:

开发一种高通量方法,以单细胞分辨率可可视化和量化mRNA拼接变体.
克服传统RNA-FISH检测内源性拼接异型的局限性.
为了使功能性基因组学屏幕能够用于基因表达和替代拼接调节.

主要方法:

开发了HiFENS (内源性前mRNA拼接异型的高通量FISH检测).
嵌入式杂交连锁反应 (HCR) 信号放大.
启用了RNA拼接变体的自动化,高通量,单细胞分析.

主要成果:

HiFENS增强了低丰度转录和短RNA目标 (例如单个外显子) 的光信号.
该方法允许可视化和对mRNA拼接变体的相对量化.
实现了高通量,单细胞分辨率用于RNA拼接变体检测.

结论:

HiFENS代表了基于FISH的高通量RNA检测的重大进步.
这种技术为功能基因组学屏幕提供了一个强大的工具.
HiFENS促进了调节基因表达和替代性mRNA前拼接的途径的发现和剖析.

关键词:

替代性的mRNA前拼接.在 FGFR2 中进行拼接.高通量成像技术的成像通过RNA成像进行RNA成像.在RNA-HCR-FISH中使用.

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RNA Splicing

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...

Alternative RNA Splicing

Alternative RNA Splicing

Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...