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This second edition details new and updated methods on circular RNA. Chapter guide readers through circular RNA purification, in silico characterization, circRNA detection, sequence validation, quantification , techniques related to gain- and loss-of-function approaches, circular RNA synthesis, split ligation, engineering, nanoparticle packaging, RNA modifications on circular RNA biogenesis, RNA translation potential, and vaccines based on circular RNAs. 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 key tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Circular RNAs, Second Edition aims to ensure successful results in the further study of this vital field.
List of contents
Purification of circular RNAs using poly(A) tailing followed by RNase R digestion.- State of the art circular RNA analytics using the circtools software suite.- Exploring circular RNA profile and expression in extracellular vesicles.- Sanger sequencing to determine the full-length sequence of circular RNAs.- Droplet digital PCR for the detection and quantification of bona fide circRNAs.- Targeted sequencing of circular RNAs for Illumina-based counting and Nanopore structure determination.- Nanopore mediated sequencing of circular RNA.- In vivo tissue-specific knockdown of circRNAs using shRNAs in Drosophila Melanogaster.- Functional circular RNA screening via RfxCas13d/BSJ-gRNA system.- Base-editor-mediated circRNA knockout by targeting predominantly back-splice sites.- Directed Circularization of a Short RNA.- Engineering Synthetic circRNAs for Efficient CNS Expression.- Functional investigation of in vitro transcribed circular RNA and formulation into Lipid nanoparticles via microfluidic mixing.- Characterising post-transcriptional modifications of circRNAs to investigate biogenesis and translation.- Native circular RNA pulldown method to simultaneously profile RNA and protein interactions.- Screening and characterization of functional circRNAs in neuronal cultures.
