Synthesis and labeling of RNA in vitro

Process Science Downstream, Bristol-Myers Squibb Company, East Syracuse, New York.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 04/2013; Chapter 4:Unit4.15. DOI: 10.1002/0471142727.mb0415s102
Source: PubMed


This unit discusses several methods for generating large amounts of uniformly labeled, end-labeled, and site-specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5' dephosphorylation and rephosphorylation, 3' terminal nucleotide addition (via ligation), site-specific RNase H cleavage directed by 2'-O-methyl RNA-DNA chimeras, and 2-piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. Curr. Protoc. Mol. Biol. 102:4.15.1-4.15.14. © 2013 by John Wiley & Sons, Inc.

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    ABSTRACT: This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3'-terminal labeling using nucleotide (5' [(32) P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good electrophoretic separation of RNA are also discussed. Curr. Protoc. Mol. Biol. 105:4.20.1-4.20.13. © 2014 by John Wiley & Sons, Inc.
    Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 02/2014; 105:4.20.1-4.20.13. DOI:10.1002/0471142727.mb0420s105
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