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

Xuan Guo; Yiqing Zhao; Hieu Nguyen; Tonghua Liu; Zhenghe Wang; Hua Lou

doi:10.3791/57889

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

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

8.4K views

⸱

DOI:

10.3791/57889

⸱

August 26th, 2018

Xuan Guo¹^,² , Yiqing Zhao¹^,³ , Hieu Nguyen¹ , Tonghua Liu² , Zhenghe Wang¹^,³ , Hua Lou¹^,³^,⁴

¹Department of Genetics and Genome Sciences, Case Western Reserve University, ²Department of Endocrinology, Dongfang Hospital of Beijing University of Chinese Medicine, ³Case Comprehensive Cancer Center, Case Western Reserve University, ⁴Center for RNA Science and Therapeutics, Case Western Reserve University

Summary

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Abstract

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Introduction

Alternative splicing (AS) is a common process that occurs during pre-mRNA maturation. In this process, an exon can be differentially included in mature mRNA. Thus, through AS, one gene can generate many mRNAs that code for different protein products. It is estimated that 92–94% of human genes undergo alternative splicing¹^,². Abnormal alternative splicing patterns resulted from genetic mutations have been linked to a large number of diseases, including amyotrophic lateral sclerosis, myotonic dystrophy, and cancer³^,⁴. It is thus crucial to investig....