Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR.
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Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR. / Tolker-Nielsen, Tim; Holmstrøm, K; Molin, S.
In: Applied and Environmental Microbiology, Vol. 63, No. 11, 1997, p. 4196-203.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR.
AU - Tolker-Nielsen, Tim
AU - Holmstrøm, K
AU - Molin, S
N1 - Keywords: Lac Operon; Polymerase Chain Reaction; RNA, Bacterial; RNA, Messenger; Salmonella typhimurium; Sensitivity and Specificity
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
M3 - Journal article
C2 - 9361404
VL - 63
SP - 4196
EP - 4203
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 11
ER -
ID: 8780564