This first book on the market covers the many new and important RNA species discovered over the past five years, explaining current methods for the enrichment, separation and purification of these novel RNAs. Building up from general principles of RNA biochemistry and biophysics, this book addresses the practical aspects relevant to the laboratory researcher throughout, while discussing the performance and potential problems of the methods discussed. An appendix contains a glossary with the important terms and techniques used in RNA analysis. By explaining the basic and working principles of the methods, the book allows biochemists and molecular biologists to gain much more expertise than by simply repeating a pre-formulated protocol, enabling them to select the procedure and materials best suited to the RNA analysis task at hand. As a result, they will be able to develop new protocols where needed and optimize and fine-tune the general purpose standard protocols that come with the purification equipment and instrumentation.
Douglas Gjerde received his PhD in analytical chemistry from Iowa State University (USA). After having worked in large and small companies for a number of years, Dr. Gjerde founded Sarasep (now merged into Transgenomic) in 1990 for the production and commercialization of nucleic acid separation technologies. He has authored and coauthored numerous articles and 4 books and holds nearly fifty patents in separation science. Lee Hoang is a graduate of the University of California, Santa Cruz (USA) where he obtained a Ph.D. in Molecular Biology in 2006. He is now an applications scientist at PhyNexus in San Jose, a company specializing on protein purification that was founded by Douglas Gjerde. David Hornby studied biochemistry at the University of Sheffield (UK). Since 1999 he is a full professor for Molecular Biology and Biotechnology at the same institution. He has also co-founded two start-up companies (Invector and Protealysis), based on novel technologies for RNA separation and stabilisation, for which he holds the patents. His current research focuses on RNAi biochemistry and proteomics technologies.
RNA EXTRACTION, SEPARATION AND ANALYSIS The need to be able to extract, manipulate and analyze RNA Using chemical tools to solve the problem of analysis of biological problems The principle of chromatography and solid phase extraction RNA Chromatography Enzymatic treatment of RNA and analysis Content and organization of this book BIOLOGICAL AND CHEMICAL RNA Why Classify RNA with biology and chemistry? Prokaryote cellular RNA Prokaryote sample type Eukaryote cellular RNA Eukaryote sample type Other Samples (Virus, Soil, Rock) Synthetic RNA RNA SEPARATION - SUBSTRATES, FUNCTIONAL GROUPS, MECHANISMS AND CONTROL Solid phase interaction The solid phase substrate and attachment of functional groups Reverse phase ion pairing separation mechanism Ion exchange separation mechanism Chaotropic denaturing interaction mechanism Hybridization / SELEX Gel Filtration RNA EXTRACTION AND ANALYSIS Transcription Translation Gene regulation Use of siRNA to investigate gene function RNA CHROMATOGRAPHY Development of RNA Chromatography RNA Chromatography instrumentation RNA Chromatography conditions Temperature modes of RNA Chromatography Comparison of gel electrophoresis and liquid chromatography Analysis of human telomerase RNA under non denaturing conditions RNA CHROMATOGRAPHY, SEPARATION AND ANALYSIS Features of RNA Chromatography Separation of double-stranded and single-stranded RNA Separation of cellular RNA species Separation of messenger RNA from total and ribosomal RNA Analysis of transfer RNA Chromatography and analysis of synthetic oligo ribonucleotides Application of RNA and DNA Chromatography in cDNA library synthesis Chromatography analyses of RT-PCR and competitive RT-PCR products Alternative splicing Differential messenger RNA display via RNA Chromatography RNA STRUCTURE-FUNCTION PROBING Definition of the Structure-function paradigm Structure determination of RNA Footprinting, model building and functional investigations APPENDIX Chromatographic Separation Equotations and Priciples for RNA Separation HPLC Instrumentation and Operation RNA Chromatographic System Cleaning and Passivation Treatment