In Vitro Splicing Assays

In vitro splicing assays represent an important technique to analyze, in detail, the basic splicing mechanisms for a pre‐mRNA of interest. The cell‐free assays, under defined conditions, have many advantages for characterizing the cis‐acting elements and trans‐acting factors responsible for the regu...

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Bibliographic Details
Published inAlternative pre‐mRNA Splicing pp. 320 - 329
Main Authors Mayeda, Akila, Krainer, Adrian R
Format Book Chapter
LanguageEnglish
Published Weinheim, Germany Wiley‐VCH Verlag GmbH & Co. KGaA 11.01.2012
Subjects
denaturing polyacrylamide gel electrophoresis
HeLa cell nuclear extract
In vitro splicing
in vitro transcription
pre‐mRNA substrate
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ISBN3527326065
9783527326068
DOI10.1002/9783527636778.ch30

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Summary:In vitro splicing assays represent an important technique to analyze, in detail, the basic splicing mechanisms for a pre‐mRNA of interest. The cell‐free assays, under defined conditions, have many advantages for characterizing the cis‐acting elements and trans‐acting factors responsible for the regulation of alternative splicing. Radiolabeled transcripts of minigenes of interest are spliced in HeLa cell nuclear extracts under various conditions, and the splicing products analyzed directly using denaturing polyacrylamide gel electrophoresis, or detected indirectly using RT‐PCR. Here, a standard method of in vitro splicing is described that has been used successfully for many years in the authors' laboratories.
ISBN:3527326065
9783527326068
DOI:10.1002/9783527636778.ch30