title: Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes.
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.
authors.affiliation: Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Annelie
authors.initials: A
authors.lastname: Pernthaler
authors.forename: Jakob
authors.initials: J
authors.lastname: Pernthaler
mesh_terms: D000818:Animals; D001483:Base Sequence; D004076:Digoxigenin / Q000737:chemistry; D004783:Environmental Microbiology; D019793:Fluorescein / Q000737:chemistry; D015870:Gene Expression; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D049879:Mytilidae / Q000382:microbiology; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics; D015347:RNA Probes / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis*
chemical_list: D015347:RNA Probes; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D010105:Oxygenases; C021555:methane monooxygenase; D004076:Digoxigenin; D019793:Fluorescein