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 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.