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.

This laboratory guide represents a growing collection of tried, tested and optimized laboratory protocols for the isolation and characterization of eukaryotic RNA, with lesser emphasis on the characterization of prokaryotic transcripts. Collectively the chapters work together to embellish the RNA story, each presenting clear take-home lessons, liberally incorporating flow charts, tables and graphs to facilitate learning and assist in the planning and implementation phases of a project. RNA Methodologies, 3rd edition includes approximately 30% new material, including chapters on the more recent technologies of RNA interference including: RNAi; Microarrays; Bioinformatics. It also includes new sections on: new and improved RT-PCR techniques; innovative 5’ and 3’ RACE techniques; subtractive PCR methods; methods for improving cDNA synthesis. * Author is a well-recognized expert in the field of RNA experimentation and founded Exon-Intron, a well-known biotechnology educational workshop center * Includes classic and contemporary techniques * Incorporates flow charts, tables, and graphs to facilitate learning and assist in the planning phases of projects

Legionnaires' disease, a pneumonia caused by the Legionella bacterium, is the leading cause of reported waterborne disease outbreaks in the United States. Legionella occur naturally in water from many different environmental sources, but grow rapidly in the warm, stagnant conditions that can be found in engineered water systems such as cooling towers, building plumbing, and hot tubs. Humans are primarily exposed to Legionella through inhalation of contaminated aerosols into the respiratory system. Legionnaires' disease can be fatal, with between 3 and 33 percent of Legionella infections leading to death, and studies show the incidence of Legionnaires' disease in the United States increased five-fold from 2000 to 2017. Management of Legionella in Water Systems reviews the state of science on Legionella contamination of water systems, specifically the ecology and diagnosis. This report explores the process of transmission via water systems, quantification, prevention and control, and policy and training issues that affect the incidence of Legionnaires' disease. It also analyzes existing knowledge gaps and recommends research priorities moving forward.

This book provides a compendium of state-of-the-art methods for the labeling, detection, and purification of RNA and RNA-protein complexes and thereby constitutes an important toolbox for researchers interested in understanding the complex roles of RNA molecules in development, signaling, and disease. Beginning with a section on in situ detection of RNA molecules using FISH techniques, the volume continues with parts exploring in vivo imaging of RNA transport and localization, imaging and analysis of RNA uptake and transport between cells, identification and analysis of RNA-binding proteins, guide RNAs in genome editing, as well as other specific analytical techniques. Written for the highly successful Methods in Molecular Biology series, 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. Authoritative and practical, RNA Tagging: Methods and Protocols serves as a vital reference for researchers looking to further the increasingly important research in RNA biology.

Early detection is essential to the control of emerging, reemerging, and novel infectious diseases, whether naturally occurring or intentionally introduced. Containing the spread of such diseases in a profoundly interconnected world requires active vigilance for signs of an outbreak, rapid recognition of its presence, and diagnosis of its microbial cause, in addition to strategies and resources for an appropriate and efficient response. Although these actions are often viewed in terms of human public health, they also challenge the plant and animal health communities. Surveillance, defined as "the continual scrutiny of all aspects of occurrence and spread of a disease that are pertinent to effective control", involves the "systematic collection, analysis, interpretation, and dissemination of health data." Disease detection and diagnosis is the act of discovering a novel, emerging, or reemerging disease or disease event and identifying its cause. Diagnosis is "the cornerstone of effective disease control and prevention efforts, including surveillance." Disease surveillance and detection relies heavily on the astute individual: the clinician, veterinarian, plant pathologist, farmer, livestock manager, or agricultural extension agent who notices something unusual, atypical, or suspicious and brings this discovery in a timely way to the attention of an appropriate representative of human public health, veterinary medicine, or agriculture. Most developed countries have the ability to detect and diagnose human, animal, and plant diseases. Global Infectious Disease Surveillance and Detection: Assessing the Challenges-Finding Solutions, Workshop Summary is part of a 10 book series and summarizes the recommendations and presentations of the workshop.

Since its invention and subsequent development nearly 20 years ago, po- merase chain reaction (PCR) has been extensively utilized to identify numerous gene probes in vitro and in vivo. However, attempts to generate complete and full-length complementary cDNA libraries were, for the most part, fruitless and remained elusive until the last decade, when simple and rapid methods were developed. With current decoding and potential application of human genome information to genechips, there are urgent needs for identification of functional significance of these decoded gene sequences. Inherent in bringing these app- cations to fruition is the need to generate a complete and full-length cDNA library for potential functional assays of specific gene sequences. Generation of cDNA Libraries: Methods and Protocols serves as a laboratory manual on the evolution of generation of cDNA libraries, covering both ba- ground information and step-by-step practical laboratory recipes for which p- tocols, reagents, operational tips, instrumentation, and other requirements are detailed. The first chapter of the book is an overview of the basics of generating cDNA libraries, which include the following: (a) the definition of a cDNA library, (b) different kinds of cDNA libraries, (c) differences between methods for cDNA library generation using conventional approaches and novel stra- gies, including reverse generation of RNA repertoires from cDNA libraries, and (d) the quality of cDNA libraries.