@article{e4a5add0bd4111dd8e02000ea68e967b,
title = "In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells.",
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.",
author = "M Lange and Tim Tolker-Nielsen and S Molin and Ahring, {B K}",
note = "Keywords: Cell Wall; DNA Primers; Digoxigenin; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Indicators and Reagents; Methanosarcina; Molecular Chaperones; Muramidase; RNA, Archaeal; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic",
year = "2000",
language = "English",
volume = "66",
pages = "1796--800",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "5",
}