Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR

Tim Tolker-Nielsen, Kim Holmstrøm, Søren Molin

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac(+) strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays, Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.
    Original languageEnglish
    JournalApplied and Environmental Microbiology
    Volume63
    Issue number11
    Pages (from-to)4196-4203
    ISSN0099-2240
    Publication statusPublished - 1997

    Fingerprint Dive into the research topics of 'Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR'. Together they form a unique fingerprint.

    Cite this