RNA Isolation and Characterization Protocols, 1998 Methods in Molecular Biology Series, Vol. 86

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.

to Isolating RNA.- Large and Small Scale RNA Preparations from Eukaryotic Cells.- An Improved Rapid Method of Isolating RNA from Cultured Cells.- Isolating RNA with the Cationic Surfactant, Catrimox-14.- RNA Extraction from Formalin-Fixed and Paraffin-Embedded Tissues.- Extraction and Purification of RNA from Plant Tissue Enriched in Polysaccharides.- Isolation of Plant Mitochondrial RNA from Green Leaves.- Extraction of RNA from Fresh and Frozen Blood.- Isolation of Total RNA from Bacteria.- Isolation of Total RNA from Tissues or Cell Lines.- Isolation of Messenger RNA.- UV Spectrophotometric Analysis of Ribonucleic Acids.- Formaldehyde Gel Electrophoresis of Total RNA.- Preparation of RNA Dot-Blots.- Nonradioactive Northern Blotting.- The Use of RNA Probes for the Analysis of Gene Expression.- Analysis of RNA by Northern Blotting Using Riboprobes.- RNA Quantitative Analysis from Fixed and Paraffin-Embedded Tissues.- Quantitative Analysis of RNA Species by PCR and Solid-Phase Minisequencing.- Preparation of Tissue Sections and Slides for mRNA Hybridization.- Detecting mRNA in Tissue Sections with Digoxigenin-Labeled Probes.- One-Tube RT-PCR with Sequence-Specific Primers.- Identification of Differentially Expressed Genes by Nonradioactive Differential Display of Messenger RNA.- Characterization of RNA Using Continuous RT-PCR Coupled with ELOSA.- Gene Expression Analysis by CD-RT-PCR Eric de Kant.- Primer Extension Analysis of mRNA.- S1 Mapping Using Single-Stranded DNA Probes.- Measurements of Rate of Transcription in Isolated Nuclie by Nuclear “Run-Off” Assay.- Transcription In Vitro Using Bacteriophage RNA Polymerases.- In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte Lysate Cell-Free System.- In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System.- The Xenopus Egg Extract Translation System.- Purification and Characterization of Viral dsRNA Genome Profiles by Crosshybridization.