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Methods Enzymol. 2005;397:352-71.

Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes.

Author information

1
Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany.

Abstract

A protocol is presented for the detection of gene expression in environmental microorganisms by means of fluorescence in situ hybridization (FISH). Messenger RNA (mRNA) is hybridized with digoxigenin (DIG)- or fluorescein (FLUOS)-labeled ribonucleotide probes. Subsequently the hybrid is detected immunochemically with a horseradish peroxidase (HRP)-labeled antibody and tyramide signal amplification (catalyzed reporter deposition, CARD). After mRNA FISH, microorganisms can be identified by rRNA FISH with oligonucleotide probes labeled either with a fluorochrome or with HRP. Sample preparation and cell permeabilization strategies for various microbial cell types are discussed. The synthesis of DIG- and FLUOS-labeled probes, as well as custom labeling of tyramides with different fluorochromes, is described. As a case study, we describe in detail mRNA FISH of the particulate methane-monooxygenase, subunit A (pmoA) in endosymbiotic bacteria from tissue sections of a marine mollusc. PmoA is used as a marker gene for methanotrophy.

PMID:
16260302
DOI:
10.1016/S0076-6879(05)97021-3
[Indexed for MEDLINE]

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