Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria
| Autor: | Rudolf Amann, Annelie Pernthaler |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Cell Membrane Permeability
Methane monooxygenase Environment Biology Polymerase Chain Reaction Applied Microbiology and Biotechnology Horseradish peroxidase chemistry.chemical_compound Ribonucleases Diethyl Pyrocarbonate Methods medicine Digoxigenin RNA Messenger Enzyme Inhibitors In Situ Hybridization Fluorescence Bacteria Ecology medicine.diagnostic_test Ribosomal RNA Proteinase K biology.organism_classification Molecular biology RNA Bacterial chemistry Biochemistry RNA Ribosomal biology.protein Lysozyme Food Science Biotechnology Fluorescence in situ hybridization |
| Zdroj: | Applied and Environmental Microbiology |
| ISSN: | 1098-5336 0099-2240 |
| Popis: | We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis -platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p -Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures. |
| Databáze: | OpenAIRE |
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