ArticleApplied and environmental microbiology2004
Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria.
Article in Applied and environmental microbiology, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers.
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Who cites it
42 citing papers in PubMed, 204 citations in OpenAlex.
- Spatial mapping of microbial communities by an integrated automation platform of sequential FISH.Cell reports methods · 2026Article
- Spatial profiling of microbial communities by sequential FISH with error-robust encoding.Nature communications · 2023Article
- Phylogenetic and functional diverse ANME-1 thrive in Arctic hydrothermal vents.FEMS microbiology ecology · 2022Article
- Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic COMicrobiome · 2022Article
- FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities.Microbial ecology · 2022Article
- Identification of housekeeping genes of Candidatus Branchiomonas cysticola associated with epitheliocystis in Atlantic salmon (Salmo salar L.).Archives of microbiology · 2022Article
- Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.Infection and immunity · 2022Article
- Comprehensive Assessment of the Virulence FactorsJournal of fungi (Basel, Switzerland) · 2021Article
- Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Bioinformatic Tools and Guidelines for the Design of Fluorescence In Situ Hybridization Probes.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Article
- The Implications of Fragmented Genomic DNA Size Range on the Hybridization Efficiency in NanoGene Assay.Sensors (Basel, Switzerland) · 2018Article
- Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia.Environmental microbiology · 2018Article
- Unravelling the Identity, Metabolic Potential and Global Biogeography of the Atmospheric Methane-Oxidizing Upland Soil Cluster α.Environmental microbiology · 2018Article
- Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH.The ISME journal · 2016Article
- A functional perspective on phenotypic heterogeneity in microorganisms.Nature reviews. Microbiology · 2015Review
- The effect of nutrients on carbon and nitrogen fixation by the UCYN-A-haptophyte symbiosis.The ISME journal · 2015Article
- Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.Applied and environmental microbiology · 2014Article
- In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry.Environmental microbiology · 2014Article
- Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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