In vivo run-on assays to monitor nascent precursor RNA transcripts

Piergiorgio Percipalle, Emilie Louvet

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Abstract

    Biochemical methods have provided mechanistic insights into the different transcription phases during which the RNA polymerase is assembled at gene promoter and becomes engaged in the elongation of nascent transcripts. Evidence that transcription takes place in specific regions of the nucleus has fuelled the need to develop assays that can be performed in living cells and provide information on the location of the specific foci, where transcription takes place. In this chapter, we describe a method that is based on the incorporation of a fluorine-conjugated uridine analogue, incorporation that can be monitored by immunofluorescence and light microscopy using specific fluorochrome- conjugated monoclonal antibodies. This assay allows direct monitoring of active transcription foci in living cells. When coupled to suitable software, the method outlined here also provides a semiquantitative approach to measure the number of active transcription foci that correlate with the proliferation state of the cell. Therefore, the assay we present here is a sensitive analytical tool to monitor the topology of transcription foci in the eukaryotic cell nucleus and to gain insight into transcription rates.

    Original languageEnglish (US)
    Title of host publicationTranscriptional Regulation
    Subtitle of host publicationMethods and Protocols
    Pages519-533
    Number of pages15
    DOIs
    StatePublished - Jan 2 2012

    Publication series

    NameMethods in Molecular Biology
    Volume809
    ISSN (Print)1064-3745

    Fingerprint

    RNA Precursors
    Fluorine
    Uridine
    Eukaryotic Cells
    DNA-Directed RNA Polymerases
    Cell Nucleus
    Fluorescent Dyes
    Fluorescence Microscopy
    Software
    Monoclonal Antibodies
    Cell Proliferation
    Light
    Genes

    Keywords

    • Confocal microscopy
    • Image processing and analysis
    • Immunofluorescence
    • Nascent RNA transcripts
    • Pre-mRNA, transcription inhibitors
    • Transcription foci

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

    Cite this

    Percipalle, P., & Louvet, E. (2012). In vivo run-on assays to monitor nascent precursor RNA transcripts. In Transcriptional Regulation: Methods and Protocols (pp. 519-533). (Methods in Molecular Biology; Vol. 809). https://doi.org/10.1007/978-1-61779-376-9_34

    In vivo run-on assays to monitor nascent precursor RNA transcripts. / Percipalle, Piergiorgio; Louvet, Emilie.

    Transcriptional Regulation: Methods and Protocols. 2012. p. 519-533 (Methods in Molecular Biology; Vol. 809).

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Percipalle, P & Louvet, E 2012, In vivo run-on assays to monitor nascent precursor RNA transcripts. in Transcriptional Regulation: Methods and Protocols. Methods in Molecular Biology, vol. 809, pp. 519-533. https://doi.org/10.1007/978-1-61779-376-9_34
    Percipalle P, Louvet E. In vivo run-on assays to monitor nascent precursor RNA transcripts. In Transcriptional Regulation: Methods and Protocols. 2012. p. 519-533. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-376-9_34
    Percipalle, Piergiorgio ; Louvet, Emilie. / In vivo run-on assays to monitor nascent precursor RNA transcripts. Transcriptional Regulation: Methods and Protocols. 2012. pp. 519-533 (Methods in Molecular Biology).
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