In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells

Marianne Lange, Tim Tolker-Nielsen, Søren Molin, Birgitte Kiær Ahring

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection system and binding of anti-digoxigenin-alkaline phosphatase conjugate, Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.
    Original languageEnglish
    JournalApplied and Environmental Microbiology
    Volume66
    Issue number5
    Pages (from-to)1796-1800
    ISSN0099-2240
    DOIs
    Publication statusPublished - 2000

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