With its complex and extensively regulated metabolism, the study of the RNA lifecycle demands tools that allow for the localization of RNAs to be observed either in an in situ setting or, preferably, under in vivo conditions. In RNA Detection and Visualization: Methods and Protocols, the best and brightest investigators provide an up-to-date and in-depth description of basic methods and protocols used for detecting and visualizing mRNAs in both fixed and live cells, from bacteria to mammals. For novices and experts alike, this mix of classic in situ hybridization and advanced live imaging techniques, cell fractionation and affinity purification procedures, and bioinformatics tools gives researchers the most complete and extensive array of research aids possible. As a volume written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, RNA Detection and Visualization: Methods and Protocols offers well-honed techniques in order to inspire researchers around the world to further our knowledge of the vital biological significance of RNA.

Methods in Enzymology: Visualizing RNA Dynamics in the Cell continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods visualizing RNA dynamics in the cell, and includes sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2, and visualization of 5S dynamics in live cells using photostable corn probe. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in visualizing RNA dynamics in the cell Contains sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2 and visualization of 5S dynamics in live cells using photostable corn probe

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.

In recent years, RNA’s structure and function has been increasingly analyzed through the utilization of experimental methods, such as RNA in situ hybridization (ISH) and electrophoretic mobility shift assays. Oligonucleotide probes are used for the detection and visualization of target RNAs for obtaining greater insight into the mechanism of gene expression and RNA function. RNA Fluorescence In-Situ Hybridization (FISH) and molecular beacons are popular tools used to detect and track endogenous RNAs. An additional tool utilized to study RNA function is the triplex forming oligonucleotide (TFO), which is a short probe that can bind to a purine-rich double stranded region within a target RNA to form a triple helix. In our study, a new probe was designed and evaluated for its efficiency in detecting a model RNA using peptide nucleic acid (PNA) TFOs. The PNA TFO probe was tested to determine the extent to which it formed a triple helix with a model RNA hairpin using two methods: 1) spectrofluorometer assays and 2) gel electrophoresis. Triplex formation was confirmed using the gel electrophoresis assay. Moreover, new theoretical probes were designed using the PinMol and TFOFinder software. Moving forward, these probes will be further tested using the experimental methodologies described in this study to evaluate their efficiency in detecting and visualizing endogenous RNA in live and fixed tissue.

Knowledge about ribonucleic acids (RNAs) and their roles has recently been expanded by extensive studies seeking to uncover novel RNAs using biochemical techniques. These techniques visualize specific RNA in situ to understanding the regulatory mechanisms of gene expression. Lately, a variety of photo labeling techniques has been developed for imaging RNA in vivo to locate and track it with high accuracy. This study focuses on fluorescence-based methods of in vivo RNA visualization. Using fluorescent light has transformed the study of micro-molecular components due to its many advantages. Molecular beacons targeting repeat expansion of RNA can differentiate between normal repeat sizes and repeat expansions in the pathogenic range. With modification and testing, molecular beacons have the potential to serve as probes for sensing repeat structure accessibility and for use in diagnostics assays. Broccoli GFP-mimic RNAs have different instability and metal ion preferences than Baby Spinach, a truncated version of the Spinach aptamer, that are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs, which likely explains their sensitivity to magnesium due to its stability and fluorescence. RNA Mango is an RNA aptamer that binds thiazole Orange (TO1) dye and its derivative with nanomolar binding affinity, unlike MS2 and Spinach systems. Mango is superior in its detection of bacterial fluorescence compared with Spinach, which requires 200uM of the fluorophore. Inserting a Mango aptamer with TO1-Biotin, a derivative of TO1, genetically into the cell can be used to monitor the expression of RNA in real time by measuring the fluorescence signal that comes from the TO1-Biotin.

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.

This book examines a wide range of techniques on RNA extraction, detection, quantification, visualization, and genome-wide profiling, from conventional methods to state-of-the-art high throughput approaches.