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Full Description
This detailed book describes some of the most recent advances and up-to-date methodologies to detect, quantify, analyze, and elucidate the biological function of different types of RNA modifications. Importantly, the methodologies and tools described herein can be applied to a wide variety of organisms and can be used to address biological and clinical questions. Beginning with a section on bioinformatics tools, the collection continues with sections on detecting RNA modifications using Nanopore direct RNA sequencing, next-generation sequencing approaches, qPCR- and molecular biology-based methods, mass spectrometry- and NMR-based methods, as well as approaches to assess kinetics, determinants, and functions of RNA modifications. Written for the highly successful Methods in Molecular Biology series style, chapters include introduction 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.
Authoritative and practical, RNA Modifications: Methods and Protocols serves as an ideal guide for those working directly in the fields of epitranscriptomics and post-transcriptional gene regulation, as well as for scientists and clinicians interested in bioinformatic tools to study RNA modifications and techniques to dissect their roles in physiology and disease.
Chapter 20 is available open access under a CC BY 4.0 license.
Contents
RNA Post-Transcriptional Modification Mapping Data Analysis Using RNA Framework.- An Informatics Pipeline for Profiling and Annotating RNA Modifications.- EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing.- Adaptation of Human Ribosomal RNA for Nanopore Sequencing of Canonical and Modified Nucleotides.- AlkAniline-Seq: A Highly Sensitive and Specific Method for Simultaneous Mapping of 7-Methyl-Guanosine (m7G) and 3-Methyl-Cytidine (m3C) in RNAs by High-Throughput Sequencing.- Transcriptome-Wide Detection of Internal N7-Methylguanosine.- miCLIP-MaPseq Identifies Substrates of Radical SAM RNA Methylating Enzyme Using Mechanistic Crosslinking and Mismatch Profiling.- Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.- Quantitative and Single Nucleotide Resolution Profiling of RNA 5-Methylcytosine.- A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs.- Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.- Relative Quantification of Residue Specific m6A RNA Methylation Using m6A-RT-QPCR.- Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease.- Analysis of Queuosine tRNA Modifications Using APB Northern Blot Assays.- Detecting ADP-Ribosylation in RNA.- Detecting Internal N7-Methylguanosine mRNA Modifications by Differential Enzymatic Digestion Coupled with Mass Spectrometry Analysis.- A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing Both Canonical and Modified Nucleotides.- Quantification of Modified Nucleosides in the Context of NAIL-MS.- A Method to Monitor the Introduction of Post-Transcriptional Modifications in tRNAs with NMR Spectroscopy.- Effects of mRNA Modifications on Translation: An Overview.- Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNAs and Stand-Alone Pseudouridine Synthases.- Investigating Pseudouridylation Mechanisms by High-Throughput In Vitro RNA Pseudouridylation and Sequencing.- Targeted RNA m6A Editing Using Engineered CRISPR-Cas9 Conjugates.
