RNA Isolation and Characterization Protocols

Ribonucleic acids are central to cellular and molecular processes and perform vital functions in both structural and functional roles. RNA molecules form the bridge between the stable genetic information contained within DNA and enzymes and proteins that carry out much of the metabolism within the cell. Many of the sites of protein synthesis, the ribosomes within the cell, are composed of these ribonucleic acids as are the tRNA molecules that deliver the amino acid building blocks to the ribosomes. Of all the RNA species, the nucleic acid intermediate, messenger RNA, is a desirable source of material to biologists, since this reflects much of, what ultimately, is translated into enzymes and proteins. In order to determine the qualitative and quantitative changes in mRNA expression, a vast number of molecular biological techniques have been developed. Key molecular methods that provide the means to initially isolate and analyze RNA molecules are the focus of this volume. In putting together this collection of protocols, we have tried to provide techniques that are most applicable and widely used. In particular, there are a number of iso- tion techniques included that have been developed, modified, or adapted to enable extraction from a variety of cell types, organisms, or subcellular organelles. Successful isolation of intact RNA is an essential starting point for any sub- quent analysis. This is why we have aimed to make this section comprehensive. The analysis of RNA is the focus of the following chapters.

Table of Contents:
Introduction to Isolating RNA Donald E. Macfarlane and Christopher E. Dahle. Large and Small Scale RNA Preparations from Eukaryotic Cells Wolfgang Uckert, Wolfgang Walther, and Ulrike Stein. An Improved Rapid Method of Isolating RNA from Cultured Cells David B. Batt, Gordon G. Carmichael, and Zhong Liu. Isolating RNA with the Cationic Surfactant, Catrimox-14 Christopher E. Dahle and Donald E. Macfarlane. RNA Extraction from Formalin-Fixed and Paraffin-Embedded Tissues Giorgio Stanta, Serena Bonin, and Rosella Perin. Extraction and Purification of RNA from Plant Tissue Enriched in Polysaccharides Shu-Hua Cheng and Jeffrey R. Seemann. Isolation of Plant Mitochondrial RNA from Green Leaves Fei Ye, Wolfgang O. Abel, and Ralf Reski. Extraction from Fresh and Frozen Blood Bimal D. M. Theophilus. Isolation of Total RNA from Bacteria John Heptinstall. Isolation of Total RNA from Tissues or Cell Lines: Visualization in Gel Tapas Mukhopadhyay and Jack A. Roth. Isolation of Messenger RNA Sian Bryant and David L. Manning Formaldehyde Gel Electrophoresis of Total RNA Sian Bryant and David L. Manning. UV Spectrophotometric Analysis of Ribonucleic Acids Ralph Rapley and John Heptinstall. Preparation of RNA Dot-Blots Rachel Hodge. Nonradioactive Northern Blotting Rainer Löw. The Use of RNA Probes for the Analysis of Gene Expression Dominique Belin. Analysis of RNA by Northern Blotting Using Riboprobes Rai Ajit K. Srivastava. RNA Quantitative Analysis form Fixed and Paraffin-Embedded Tissues Giorgio Stanta, Serena Bonin, and René Utrera. Quantitative Analysis of RNA Species by PCR and Solid-Phase Minisequencing Anu Suomalainen and Ann-Christine Syvänen. Preparation of Tissue Sections and Slides for mRNA Hybridization Giorgio Terenghi. Detecting mRNA in Tissue Sections with Digoxigenin-Labeled Probes Giorgio Terenghi . One-Tube RT-PCR with Sequence-Specific Primers Ulrich Pfeffer Identification of Differentially Expressed Genes by Nonradioactive Differential Display of Messenger RNA Thomas C. G. Bosch and Jan U. Lohmann. Characterization of RNA Using Continuous RT-PCR Coupled with ELISA François Mallet, Guy Oriol, and Bernard Mandrand. Gene Expression Analysis by CD-RT-PCR Eric de Kant. Primer Extension Analysis of mRNA Maggie Walmsley, Mark Leonard, and Roger Patient. S1 Mapping Using Single-Stranded DNA Probes Stéphane Viville and Roberto Mantovani. Measurements of Rate of Transcription in Isolated Nuclei by Nuclear 'Run-Off' Assay Rai Ajit Srivastava and Gustav Schonfeld. Transcription In Vitro Using Bacteriophage RNA Polymerases Elaine T. Schenborn. In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte Lysate Cell-Free System Louise Olliver and Charles D. Boyd. In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System Louse Olliver, Ann Grobler-Rabie, and Charles D. Boyd. The Xenopus Egg Extract Translation System Glenn M. Matthews and Alan Colman. Purification and Characterization of Viral dsRNA Genome Profiles by Crosshybridization Lesley-Ann Martin and Peter P. C. Mertens. Index.