Alternative Splicing Methods and Protocols

This detailed volume collects commonly used and cutting-edge methods to analyze alternative splicing, a key step in gene regulation. After an introduction of the alternative splicing mechanism and its targeting for therapeutic strategies, the book continues with techniques for analyzing alternative splicing profiles in complex biological systems, visualizing and localizing alternative spliced transcripts with cellular and sub-cellular resolution, probing regulators of alternative splicing, as well as assessing the functional consequences of alternative splicing. Written for the highly successful Methods in Molecular Biology series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. 

Alternative Splicing in Human Biology and Disease.- Targeting Alternative Splicing for Therapeutic Interventions.- An Optimized Protocol for the Mapping of Cell Type-Specific Ribosome-Associated Transcript Isoforms from Small Mouse Brain Regions.- FACS-Based Neuronal Cell Type-Specific RNA Isolation and Alternative Splicing Analysis.- Quantitative Measurement of Alternatively Spliced RNA Isoform Levels.- Analysis of Splicing Regulation by Third-Generation Sequencing.- Computational Analysis of Alternative Splicing Using VAST-TOOLS and the VastDB Framework.- Identification and Quantification of Microexons Using Bulk and Single-Cell RNA-Seq Data.- Functional Annotation of Custom Transcriptomes.- Design, Labeling, and Application of Probes for RNA smFISH.- BaseScope™ Approach to Visualize Alternative Splice Variants in Tissue.- In Situ Imaging of mRNA Splicing Variants by SpliceRCA.- Two-Color Fluorescent Reporters for Analysis of Alternative Splicing.- Quantitative Detection of Protein Splice Variants by Selected Reaction Monitoring (SRM) Mass Spectrometry.- Probing the Interactions of Splicing Regulatory Small Molecules and Proteins with U1 snRNP Using NMR Spectroscopy.- Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention.- Tethered Function Assays to Elucidate the Role of RNA-Binding Proteins.- Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo.- Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals.