What is Stellaris RNA FISH?

Stellaris RNA FISH: Detection, Localization, and Quantification of RNA at the (Sub-)Cellular Level

Stellaris® RNA FISH (fluorescence in situ hybridization) is a RNA visualization method that allows simultaneous detection, localization, and quantification of individual RNA molecules at the sub-cellular level in fixed samples using widefield or confocal fluorescence microscopy. A set of Stellaris RNA FISH Probes comprises multiple oligonucleotides with different sequences each with a fluorescent label that collectively bind along the same target transcript to produce a punctate signal.

The Stellaris RNA FISH technology follows a simple protocol, does not use exotic reagents, is inexpensive and platform-independent. It also offers same-day results, and is versatile both in terms of sample type and application. Stellaris RNA FISH probes may even be labeled with several different dyes, to enable simultaneous multiplex detection of different RNA targets. Finally, scientists can address the stochastic nature of gene expression and visualize RNA through direct detection without isolation, purification, and amplification.

Stellaris Protocol

Stellaris RNA FISH Protocol

The Stellaris RNA FISH protocol is comparatively simple, and consists of four steps, as shown in the image below. No exotic reagents are required, and the entire process can be completed in less than a day.

The Stellaris RNA FISH Method

Step 1: Prepare Sample
The sample is grown on or adhered, or sectioned onto a #1 coverglass and permeabilized with 70% ethanol. Slight variations in sample preparation between different organisms and sample types are covered in the online protocols. No protease is required.
Step 2: Hybridize Probes
For most samples, hybridization can be completed in 4 hours at +37 °C in a generic laboratory incubator.
Step 3: Wash Sample
After hybridization, wash buffers with short incubation periods are used to remove excess probes. The total time for this step is 1 – 1.5 hours.
Step 4: Image Sample
At this point, the sample can be imaged using a standard fluorescence microscope. We recommend using a widefield fluorescence microscope though confocal microscopy can also be used with Stellaris RNA FISH probes.

Stellaris RNA FISH Probes are sold under license from Rutgers, The State University of New Jersey, and may be used under its patent rights for Research Use Only.

Sensitivity & Specificity

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The large number of probes in a Stellaris RNA FISH assay ensures both high sensitivity and specificity. The inherent redundancies of this direct detection method minimizes the possibility of false negative and false positive signals. Positive signal identified from the combined localized fluorescence of multiple probes. Off-target binding of single probes generates only weak and diffuse fluorescence, well below the threshold for detection of the specifically-targeted mRNAs. Furthermore, Stellaris RNA FISH probes can bind to partially degraded target mRNAs making them well-suited for formalin-fixed, paraffin-embedded (FFPE) tissue samples. 

Stellaris RNA FISH Probes are sold under license from Rutgers, The State University of New Jersey, and may be used under its patent rights for Research Use Only.

Multiplexing

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Stellaris ENA FISH probes are available with a wide variety of fluorophores, including Biosearch Technologies’ CAL Fluor® and Quasar® dyes and can be used in a multiplex assay. The dye choices in a multiplex assay are largely determined by the filters available to a researcher, but it is certainly possible to design an assay including a DAPI nuclear stain and three Stellaris RNA FISH probe sets labeled with reporter dyes with minimal spectral overlap.

Multiplex assays can be used to identify a correlation in expression levels between multiple genes, run a control probe set of known expression with a target simultaneously, or simply save time over running the assays one at a time. For human cells, we offer inventoried probe sets suitable for use as positive controls. These foundation gene probe sets are labeled with Quasar® 570 dye.

Stellaris RNA FISH can also be combined with existing technologies such as qPCR, DNA FISH, IHC (immunohistochemistry), and western blotting to provide complementary information.

Stellaris RNA FISH Probes are sold under license from Rutgers, The State University of New Jersey, and may be used under its patent rights for Research Use Only.

Resources

Stellaris RNA FISH Probes are sold under license from Rutgers, The State University of New Jersey, and may be used under its patent rights for Research Use Only.

How to Order

Ordering Information

Go to our Stellaris RNA FISH product page to find ordering information for Stellaris Buffers and Custom, DesignReady, and ShipReady Assays.

Stellaris RNA FISH Probes are sold under license from Rutgers, The State University of New Jersey, and may be used under its patent rights for Research Use Only.

Stellaris RNA FISH product page