RNA-Protein Interaction Protocols

Volume 118 of the series Methods in Molecular Biology™ pp 315-321

Mammalian In Vitro Splicing Assays

  • Akila MayedaAffiliated withDepartment of Biochemistry and Molecular Biology, University of Miami School of Medicine
  • , Adrian R. KrainerAffiliated withCold Spring Harbor Laboratory

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Splicing reactions are typically carried out using nuclear extracts, S100 extracts complemented with SR proteins, or partially purified fractions derived from the crude extracts. The extract preparation procedures are described in Chapter 24. Extracts derived from HeLa cells are used most commonly (see Note 1). The pre-mRNA substrates are usually prepared by in vitro runoff transcription with a bacteriophage polymerase (see Chapter 1). The intermediates and products of splicing are most conveniently visualized by urea/polyacrylamide gel electrophoresis (urea-PAGE) and autoradiography, which requires the use of labeled pre-mRNA substrate. The protocols provided here are based on ref. 1, with modifications introduced in refs. 2,3, and have been routinely used in our laboratory for many years.