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Highly skilled experimentalists provide a comprehensive series of commonly used, as well as specialized, techniques for analyzing how proteins and RNA interact. Richly detailed and readily reproducible, these methods enable researchers to analyze the structural details of an RNA-protein interaction, to determine in detail what parts of the protein and RNA are in close contact, and to isolate RNP complexes from cells. There are also in vitro and in vivo methods and protocols for assaying the effects of proteins and RNP complexes on mRNA metabolism. The methods assume only a knowledge of basic molecular biology, biochemistry, and cell culture, and can be readily adapted to other systems. RNA-Protein Interaction Protocols offers a wide-ranging collection of up-to-date experimental methods that will assure productive and reproducible results for all investigators in the field.
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Methods in Molecular Biology TM VOLUME 118 RNA–Protein Interaction Protocols Edited by Susan R. Haynes HUMANA PRESS Labeling and Purification of RNA 1 1 Labeling and Purification of RNA Synthesized by In Vitro Transcription Paul A. Clarke 1. Introduction The problems of isolating sufficient quantities of rare RNAs for detailed biochemical analysis can be circumvented by synthesis of the desired RNA in vitro (1–3). Early methods of in vitro transcription included the use of eukaryotic cell extracts or Escherichia coli RNA polymerase to transcribe DNA templates containing the appropriate promoter. Ideally, however, the optimal in vitro transcription system should require simple buffer components without need for preparation of extracts and should precisely initiate and terminate transcription at definable sites. In vitro bacteriophage transcription systems fulfill these criteria. Single-stranded RNA of the desired sequenc