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 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.

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.

The aim of molecular diagnostics is preferentially to detect a developing disease before any symptoms appear. There has been a significant increase, fueled by technologies from the human genome project, in the availability of nucleic acid sequence information for all living organisms including bacteria and viruses. When combined with a different type of instrumentation applied, the resulting diagnostics is specific and sensitive. Nucleic acid-based medical diagnosis detects specific DNAs or RNAs from the infecting organism or virus and a specific gene or the expression of a gene associated with a disease. Nucleic acid approaches also stimulate a basic science by opening lines of inquiry that will lead to greater understanding of the molecules at the center of life. One can follow Richard Feynman’s famous statement “What I cannot create, I do not understand.”

Here is the most complete guide available to the isolation, analysis, and synthesis of RNA. It covers everything researchers and laboratory workers need to know about the study of gene expression via RNA analysis-from the theory behind the methods, to actual problem-solving techniques. Step-by-step protocols are presented for each method. A careful presentation of the experimental formalities of these protocols enables specialists and nonspecialists alike to implement the methods easily in the laboratory. Each protocol is accompanied by the theoretical background underlying the experimental procedure and most chapters contain illustrations of typical results and troubleshooting tips. A Laboratory Guide to RNA offers a straightforward detailed account of experimental procedures, ranging from the isolation of RNA from a variety of cell and tissue types, detection analysis, and quantitation using a range of strategies, to large- and small-scale synthesis of RNA. This unique guide not only covers established procedures such as RNA blotting and nuclease protection, but also the latest protocols for quantitative PCR and differential display. Protocols addressing in situ hybridization are highlighted in an eight-page, full-color section that illustrates the power of the technique for detection of gene expression in tissues and whole organisms. Featuring contributions from leading research laboratories and the biotechnology field, A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis provides all the methods required for RNA analysis. It is the ideal laboratory guide for research scientists, graduate students, and lab personnel who need a solid reference on the analysis of gene expression at the RNA level.

As our understanding of molecular biology continues to develop, both general improvements and specialized tools are needed for the progression of RNA detection and analysis. This thesis describes three distinct projects within the broad goal of expanding the techniques available for better interrogation of RNA. Chapter II illustrates the development of a new technique for microRNA detection. MicroRNAs are a class of small regulatory RNAs present in eukaryotes. MicroRNAs pose a challenge for detection because of their small size and sequence homology, but have drawn increasing interest due the predicted prevalence of their gene targets and links between their expression levels and cancer. The development of an isothermal detection strategy is described which incorporates two nucleic acid templated steps, two amplification steps, and fluorescent detection. Up to 3000-fold amplification can be obtained, and 200 pM quantities of let-7a microRNA and above can be detected reliably. In Chapter III, two distinct approaches are taken for the detection of N(6)-methyladenosine, the most common modification in RNA: harnessing base-pairing selectivity, and developing selective reactions. A successful method was developed that harnessed the inherently higher efficiency of a commercially-available polymerase, Tth DNA polymerase, when pairing T with adenosine as opposed to N(6)-methyladenosine. Kinetic analysis showed that the selectivity of the enzyme ranged from 4-18-fold, depending on sequence context. Detection of m6A in ribosomal RNA and in a highly-expressed messenger RNA are demonstrated. The final chapter focuses on a mechanistic approach for enhancing RNA recovery after formaldehyde treatment using an organocatalyst. Mass spectrometry and gel electrophoresis showed an enhanced rate of adduct reversal in the presence of the catalyst, and effects of temperature, buffer, and pH were also analyzed. The results from monomeric and oligomeric test systems have subsequently been applied to recovery of RNA from fixed samples, showing ~2-fold enhancement in recovered RNA due to the catalyst and up to 25-fold enhancement when the catalyst was used in conjunction with optimized time and temperature.

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods. Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies. Provides state-of-the-art PCR methods for amplification, quantitation, in situ hybridization, and multiplex reactions Goes beyond PCR with protocols for 3SR, NASBA, LCR, SDA, and LAT Covers important virus detection methods such as in situ hybridization; Southern, dot, and slot blots; branched chain signal amplification; and chemiluminescence Includes quality control information crucial in research and clinical laboratories Most chapters are written by the inventors and principal developers of the methodologies Includes color plates, 77 figures, and 18 tables