In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells.
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.
Lange
M
M
Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Tolker-Nielsen
T
T
Molin
S
S
Ahring
B K
BK
D002473:Cell Wall / Q000502:physiology; D017931:DNA Primers; D004076:Digoxigenin; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D007202:Indicators and Reagents; D017020:Methanosarcina / Q000235:genetics*; D018832:Molecular Chaperones / Q000235:genetics; D009113:Muramidase; D019642:RNA, Archaeal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D007202:Indicators and Reagents; D018832:Molecular Chaperones; D019642:RNA, Archaeal; D012333:RNA, Messenger; D009113:Muramidase; C063842:dnaK protein, E coli; D004076:Digoxigenin