Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine

  • Methods Mol Biol. 2017:1648:169-176. doi: 10.1007/978-1-4939-7204-3_13.
Angela Garibaldi  1 ,  Francisco Carranza  1 ,  Klemens J Hertel  2
Affiliations
  • 1. Department of Microbiology and Molecular Genetics, University of California, Irvine, B233 Med Sci I, Irvine, CA, 92697-4025, USA.
  • 2. Department of Microbiology and Molecular Genetics, University of California, Irvine, B233 Med Sci I, Irvine, CA, 92697-4025, USA. [email protected].
PMID: 28766297 DOI: 10.1007/978-1-4939-7204-3_13
Abstract

Isolation of newly transcribed RNA is an invaluable approach that can be used to study the dynamic life of RNA in cellulo. Traditional methods of whole-cell RNA extraction limit subsequent gene expression analyses to the steady-state levels of RNA abundance, which often masks changes in RNA synthesis and processing. This chapter describes a methodology with low cytotoxicity that permits the labeling and isolation of nascent pre-mRNA in Cell Culture. The resulting isolate is suitable for use in a series of downstream applications aimed at studying changes in RNA synthesis, processing, or stability.

Keywords
4-Thiouridine; 4sU; 4sU-seq; Decay; Mammalian cells; Metabolic labeling; Nascent RNA; Nascent pre-mRNA; Transcription; mRNA processing.
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