M. Lange et al., In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells, APPL ENVIR, 66(5), 2000, pp. 1796-1800
An in situ reverse transcription-PCR protocol for detecting specific mRNA i
n Methanosarcina mazei S-6 is described. This method allowed us to detect h
eat shock-induced increases in the intracellular levels of the transcript o
f the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed
cells were permeabilized by a thermal cycling procedure or by lysozyme trea
tment, and the cellular DNA was removed with DNase. The cells were subjecte
d to a seminested reverse transcription-PCR protocol in which a digoxigenin
-labeled primer was used. Detection of the reporter molecule was based on t
he 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection
system and binding of anti-digoxigenin-alkaline phosphatase conjugate, Fluo
rescence in permeabilized cells increased after a heat shock compared to fl
uorescence in non-heat-shocked cells, and the increase corresponded to an i
ncrease in the level of the dnaK transcript.