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This detailed volume assembles a number of the most commonly used and state-of-the-art methodologies in the revolutionary field of mRNA processing in order to aid researchers in choosing the best method for their specific problems. Given that the vast majority of alternative mRNA processing events has yet to be explored, these techniques promise to breathe new life into the myriad new ideas currently underway. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Practical and easy to use, mRNA Processing: Methods and Protocols seeks to aid scientists venturing into what's certain to be another explosive period of growth in this dynamic field of study.
The chapter 'Robust, Cost-EffectiveProfiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP)' is published open access under a CC BY 4.0 license at link.springer.com.
List of contents
Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction.- Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.- Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).- In Vitro Modulation of Endogenous Alternative Splicing Using Splice-Switching Antisense Oligonucleotides.- Purification of mRNA Processing Complexes Using an RNA Affinity Approach.- Polyadenylation Site-Based Analysis of Transcript Expression by 3'READS+.- Comprehensive Identification of mRNA Polyadenylation Sites by PAPERCLIP.- Microinjection and Fluorescence In Situ Hybridization Assay for Studying mRNA Export in Mammalian Cells.- Methods for the Detection of Adenosine-to-Inosine Editing Events in Cellular RNA.- Using RNA-Seq to Discover Genetic Polymorphisms that Produce Hidden Splice Variants.- Inducible Expression of Eukaryotic Circular RNAs from Plasmids.- Methods for Extraction of RNA, Proteins, or Protein Complexes from Subcellular Compartments of Eukaryotic Cells.- Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine.- Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-End Crosslinking and Immunoprecipitation (seCLIP).- Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae.- A Cell-Based High-Throuput Method for Identifying Modulators of Alternative Splicing.- Genome-Wide RNAi Screens for RNA Processing Events in Drosophila Melanogaster S2 Cells.- Assaying RNA Structure Inside Living Cells with SHAPE.
Summary
This detailed volume assembles a number of the most commonly used and state-of-the-art methodologies in the revolutionary field of mRNA processing in order to aid researchers in choosing the best method for their specific problems.
