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

Appl Environ Microbiol. 2000 May;66(5):1796-800. doi: 10.1128/aem.66.5.1796-1800.2000.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Wall / physiology
  • DNA Primers
  • Digoxigenin
  • Escherichia coli Proteins*
  • HSP70 Heat-Shock Proteins / genetics*
  • Indicators and Reagents
  • Methanosarcina / genetics*
  • Molecular Chaperones / genetics
  • Muramidase
  • RNA, Archaeal / genetics
  • RNA, Messenger / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Transcription, Genetic*

Substances

  • DNA Primers
  • Escherichia coli Proteins
  • HSP70 Heat-Shock Proteins
  • Indicators and Reagents
  • Molecular Chaperones
  • RNA, Archaeal
  • RNA, Messenger
  • Muramidase
  • dnaK protein, E coli
  • Digoxigenin