GeneFISH

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GeneFISH is a molecular tool that is based on Fluorescence in situ Hybridization (FISH), a method routinely used in our labs. For detailed information on the FISH technology, please refer to the 'FISH' pages at our website https://www.arb-silva.de/fish-probes/.

Gene­FISH combines the detection of specific genes and ribosomal RNA (rRNA) at the single cell level. While the presence of a certain gene is indicative of a specific functional trait, the rRNA allows for the taxonomic affiliation of the microorganism and thus, geneFISH enables us to directly link a potential metabolic function to microbial taxonomy. We target the gene of interest with dsDNA polynucleotide probes labeled multiple times with fluorophores and consecutively the rRNA with specific oligonucleotide probe(s) in two separate hybridizations.

To allow the visualization and quantification of genes at the single cell level with geneFISH, it is crucial to have: (i) efficient labeling of the polynucleotide gene probes and (ii) high-resolution microscopy to discriminate single dot-like gene signals.

 

Read more about geneFISH here: GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms

GeneFISH: gene + rRNA detection
With geneFISH the rRNA and the genes are detected in two consecutive steps: The microbial cell identity is determined with a CARD-FISH step which targets the RNA and the genes are targeted in an additional step with digoxigenin labeled polynucleotide probes and a combined antibody-CARD signal amplification step.
© Barrero-Canosa / MPI-MM

PhageFISH

The geneFISH methodology has been expanded to detect single virus particles specifically within host cells.


Read more about phageFISH here: Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses.

Burst of Pseudoalteromonas cells
Pseudoalteromonas cells (green) are about to burst from viral infection (red). Structured illumination microscopy image taken with the ELYRA PS.1 system (Zeiss).
© Barrero-Canosa / MPI-MM

Direct geneFISH

In contrast to previously published geneFISH protocols, direct geneFISH provides a protocol that allows the simultaneous hybridization of the gene and the rRNA and is therefore much faster and preserves the cell morphology and specific signals better. In combination with super-resolution microscopy geneFISH allows the sub-cellular localization of both rRNA and individual genes and it can be used for the quan­ti­fic­a­tion of per-cell gene copy num­bers. For this either the individual genes per cells are counted in structured illumination microscopy (SIM) images or the sig­nal in­tens­ity of the stained genes has to be quan­ti­fied within single cells. The Ilastik soft­ware (v. 0.5, in­cluded in the Cell­Pro­filer pack­age) for cell de­tec­tion and seg­ment­a­tion and the im­age pro­cessing pipeline within the Cell Pro­filer soft­ware (http://cellprofiler.org/download.html) can help in the process of sig­nal in­tens­ity meas­ure­ments.

A demo version of the pipeline can be found here:

Zip File for the gene signal quantification pipeline

 

Read more about direct geneFISH here: Direct-geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms

E. coli with single copy gene (red), DNA (blue) and rRNA (green).
E. coli cells with single copy genes stained in red, DNA in blue and the ribosomal RNA in green. Structured illumination microscopy image taken with the ELYRA PS.1 system (Zeiss).
© Barrero-Canosa / MPI-MM

Polynucleotide probe design

We developed a concept and software (PolyPro) for rational design of polynucleotide probe mixes to identify particular genes in defined taxa.

PolyPro consists of three modules: a GenBank Taxonomy Extractor (GTE), a Polynucleotide Probe Designer (PPD) and a Hybridization Parameters Calculator (HPC).

Find the software here for download:

The PolyPro is a freeware which runs on Windows platforms.

(Additional software for extraction of ZIP archive necessary).

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