In Situ Reverse Transcription-PCR for Monitoring Gene Expression in Individual Methanosarcina mazei S-6 Cells

AUTOR(ES)

Lange, Marianne

FONTE

American Society for Microbiology

RESUMO

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.

Documentos Relacionados

• Physiological States of Individual Salmonella typhimurium Cells Monitored by In Situ Reverse Transcription-PCR
• Immunochemical differences among Methanosarcina mazei S-6 morphologic forms.
• Detection of Human Herpesvirus 6 by Reverse Transcription-PCR
• Quantification of Clostridium botulinum Toxin Gene Expression by Competitive Reverse Transcription-PCR
• Relative Neurotoxin Gene Expression in Clostridium botulinum Type B, Determined Using Quantitative Reverse Transcription-PCR