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Applied and Environmental Microbiology, May 2000, p. 1796-1800, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Marianne Lange,1 Tim Tolker-Nielsen,2 Søren Molin,2 and Birgitte K. Ahring1,3,*

Department of Biotechnology1 and Department of Microbiology,2 Technical University of Denmark, DK-2800 Lyngby, Denmark, and School of Engineering and Applied Sciences, University of California at Los Angeles, Los Angeles, California3

Received 10 May 1999/Accepted 2 February 2000

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.

* Corresponding author. Mailing address: Institute of Biotechnology, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: (45) 45 25 61 83. Fax: (45) 45 88 32 76. E-mail: bka{at}ibt.dtu.dk.

Applied and Environmental Microbiology, May 2000, p. 1796-1800, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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