In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain temporal and spatial information about gene expression and genetic loci. While the basic workflow of ISH is similar to that of blot hybridizations—the nucleic acid probe is synthesized, labeled, purified, and annealed with the specific target—the difference is the greater amount of information gained by visualizing the results within the tissue.
There are two basic ways to visualize your RNA and DNA targets in situ—fluorescence (FISH) and chromogenic (CISH) detection. Characteristics inherent in each method of detection (see table below) have made FISH and CISH useful for very distinct applications. While both use a labeled, target-specific probe that is hybridized with the sample, the instrumentation used to visualize the samples is different for each method. Here we highlight the differences and the advantages of each method.
|Technique||Instrument/ visualization method||Primary advantage||Primary application|
|CISH||Bright-field microscopy||Ability to view the CISH signal and tissue morphology simultaneously||Molecular pathology diagnostics|
|DNA-FISH||Fluorescence microscopy||Multiplexible: visualize multiple targets in the same sample||Gene presence, copy number, and location; mutation analysis|
|RNA-FISH||Fluorescence microscopy, HCS, and flow cytometry||Multiplexible: visualize multiple targets in the same sample||Gene expression, RNA temporal and spatial localization|
Multiplexing fluorescence in situ hybridization (FISH) enables you to assay multiple targets simultaneously and visualize co-localization within a single specimen. Using spectrally distinct fluorophore labels for each different hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns in a single specimen, with multicolor visual display.
Invitrogen ViewRNA and PrimeFlow assays are a direct fluorescence RNA ISH method that uses branched DNA signal amplification (bDNA) for detection for specific signal. Multiplexing of RNA targets is possible by using independent but compatible signal amplification systems. The fluorescence RNA in situ hybridization assay has four main steps: sample preparation, target hybridization, signal amplification, and detection. This methodology results in greater specificity, lower background, and higher signal-to-noise ratios.
For Research Use Only. Not for use in diagnostic procedures.