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Full Description
This detailed volume explores techniques for researching the diverse and specialized mechanisms for mRNA degradation, both in the cytoplasm and the nucleus. From classical methods for studying RNA degradation at the single RNA level to the latest transcriptome-wide approaches involving long-read sequencing and metabolic labeling, this book focuses on methods for eukaryotic models, such as procedures for studying deadenylation, decapping and exoribonuclease activity, assessing RNA decay rate, characterizing RNA degradation intermediates, RNA-proteins interactions, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, mRNA Decay: Methods and Protocols provides both new and experienced RNA researchers with an inspiring collection of protocols to prompt further investigation of these vital degradation pathways.
Contents
Minimal Perturbation Analysis of mRNA Degradation Rates with Tet Off and RT qPCR.- Northern Blotting: Protocols for Radioactive and Non-Radioactive Detection of RNA.- RNA Blotting by Electrotransfer and RNA Detection.- Dynamic Evolution of Poly-A Tail Lengths Visualized by RNAse H Assay and Northern Blot Using Non-Radioactive Probes in Yeast.- Assessing Deadenylation Activity by Polyacrylamide Gel Electrophoresis Using a Fluorescent RNA Substrate.- Studying Exoribonuclease Activity Using Fluorescence Anisotropy Assay.- Purification of Enzymatically Active Xrn1 for Removal of Non-Capped mRNAs from In Vitro Transcription Reactions and Evaluation of mRNA Decapping Status In Vivo.- Production of Fully Capped mRNA for Transfection into Mammalian Cells: A Protocol for Enzymatic Degradation of Uncapped Transcripts after In Vitro mRNA Synthesis.- Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5'Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.- RNA Decay Assay: 5-Ethynyl-Uridine Labeling and Chasing.- Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.- Measurement of rRNA Synthesis and Degradation Rates by 3H-Uracil Labeling in Yeast.- A Method for Rapid Inducible RNA Decay.- reCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast.- Snapchat Moments of the Cell Revealed by UV-Crosslinking and RNA Co-Immunoprecipitation of Transient RNA-Protein Complexes.- Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.- Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells.- Concurrent Profiling of Localized Transcriptome and RNA Dynamics in Neurons by Spatial SLAMseq.- Analysis of Cytoplasmic RNA Decay Targets Using the Auxin Degron System.- Detection of Nuclear RNA Decay Intermediates Using a Modified Oxford Nanopore RNA Sequencing Strategy.- Defining the True Native Ends of RNAs at Single-Molecule Level with TERA-Seq.- Global Profiling and Analysis of 5' Monophosphorylated mRNA Decay Intermediates.- SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.
