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