RNA Detection: Methods and Protocols

Description

This volume introduces different concepts and methods of detecting RNA in biological material in a variety of model systems. The chapters in this book discuss methods that will answer numerous biological questions that arise in the study of RNAs. Some of the topics covered in this book are single mRNA molecule detection in embryos and neurons; detection of mRNA and associated molecules by ISH-IEM on frozen sections; optimizing molecular beacons for intracellular analysis of RNA; imaging translation dynamics of single mRNA molecules in live cells; preparation of high-throughput sequencing libraries; and capturing RNA binding proteins in embryos and in cell-culture. 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. Cutting-edge and comprehensive, RNA Detection: Methods and Protocols is a valuable resource for novel and experiences scientist in the expanding field of RNAs. Front Matter ....Pages i-xiv The Secret Life of RNA: Lessons from Emerging Methodologies (Caroline Medioni, Florence Besse)....Pages 1-28 Quantification of 2′-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol) (Lilia Ayadi, Yuri Motorin, Virginie Marchand)....Pages 29-48 Identifying the m6A Methylome by Affinity Purification and Sequencing (Phillip J. Hsu, Chuan He)....Pages 49-57 PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution (Zhipeng Lu, Jing Gong, Qiangfeng Cliff Zhang)....Pages 59-84 Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo (Toshiaki Shigeoka, Jane Jung, Christine E. Holt, Hosung Jung)....Pages 85-94 LCM-Seq: A Method for Spatial Transcriptomic Profiling Using Laser Capture Microdissection Coupled with PolyA-Based RNA Sequencing (Susanne Nichterwitz, Julio Aguila Benitez, Rein Hoogstraaten, Qiaolin Deng, Eva Hedlund)....Pages 95-110 Spatial Transcriptomics: Constructing a Single-Cell Resolution Transcriptome-Wide Expression Atlas (Kaia Achim, Hernando Martínez Vergara, Jean-Baptiste Pettit)....Pages 111-125 Single mRNA Molecule Detection in Drosophila (Shawn C. Little, Thomas Gregor)....Pages 127-142 Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos (L. Carine Stapel, Coleman Broaddus, Nadine L. Vastenhouw)....Pages 143-162 Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction (Joshua S. Titlow, Lu Yang, Richard M. Parton, Ana Palanca, Ilan Davis)....Pages 163-175 Detection of mRNA and Associated Molecules by ISH-IEM on Frozen Sections (Catherine Rabouille)....Pages 177-186 Hybridization Chain Reaction for Direct mRNA Detection Without Nucleic Acid Purification (Yao Xu, Zhi Zheng)....Pages 187-196 In Situ Detection of MicroRNA Expression with RNAscope Probes (Viravuth P. Yin)....Pages 197-208 Padlock Probes to Detect Single Nucleotide Polymorphisms (Tomasz Krzywkowski, Mats Nilsson)....Pages 209-229 Quantifying Gene Expression in Living Cells with Ratiometric Bimolecular Beacons (Yantao Yang, Mingming Chen, Christopher J. Krueger, Andrew Tsourkas, Antony K. Chen)....Pages 231-242 Optimizing Molecular Beacons for Intracellular Analysis of RNA (Mingming Chen, Yantao Yang, Christopher J. Krueger, Antony K. Chen)....Pages 243-257 Live Imaging of Nuclear RNPs in Mammalian Complex Tissue with ECHO-liveFISH (Dan Ohtan Wang)....Pages 259-272 In Vivo Visualization and Function Probing of Transport mRNPs Using Injected FIT Probes (Jasmine Chamiolo, Imre Gaspar, Anne Ephrussi, Oliver Seitz)....Pages 273-287 Visualizing RNA in Live Bacterial Cells Using Fluorophore- and Quencher-Binding Aptamers (Murat Sunbul, Ankita Arora, Andres Jäschke)....Pages 289-304 Method for Imaging Live-Cell RNA Using an RNA Aptamer and a Fluorescent Probe (Shin-ichi Sato, Kenji Yatsuzuka, Yousuke Katsuda, Motonari Uesugi)....Pages 305-318 RNA Live Imaging in the Model Microorganism Ustilago maydis (Sabrina Zander, Kira Müntjes, Michael Feldbrügge)....Pages 319-335 Real-Time Fluorescence Imaging of Single-Molecule Endogenous Noncoding RNA in Living Cells (Hideaki Yoshimura, Takeaki Ozawa)....Pages 337-347 Live Imaging of mRNA Synthesis in Drosophila (Hernan G. Garcia, Thomas Gregor)....Pages 349-357 Imaging Newly Transcribed RNA in Cells by Using a Clickable Azide-Modified UTP Analog (Anupam A. Sawant, Sanjeev Galande, Seergazhi G. Srivatsan)....Pages 359-371 Detection of the First Round of Translation: The TRICK Assay (Franka Voigt, Jan Eglinger, Jeffrey A. Chao)....Pages 373-384 Imaging Translation Dynamics of Single mRNA Molecules in Live Cells (Suzan Ruijtenberg, Tim A. Hoek, Xiaowei Yan, Marvin E. Tanenbaum)....Pages 385-404 Systematic Detection of Poly(A)+ RNA-Interacting Proteins and Their Differential Binding (Miha Milek, Markus Landthaler)....Pages 405-417 Isolation and Characterization of Endogenous RNPs from Brain Tissues (Rico Schieweck, Foong yee Ang, Renate Fritzsche, Michael A. Kiebler)....Pages 419-426 Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein–RNA Interactions (Christopher R. Sibley)....Pages 427-454 RNA Tagging: Preparation of High-Throughput Sequencing Libraries (Christopher P. Lapointe, Marvin Wickens)....Pages 455-471 RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells (Colleen A. McHugh, Mitchell Guttman)....Pages 473-488 Back Matter ....Pages 489-492