Stellaris RNA FISH
Stellaris® RNA FISH: Detection, Localization, and Quantification of RNA at the Cellular LevelWhat is Stellaris RNA FISH?
Stellaris 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 fluorescence microscopy. A set of Stellaris 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 FISH technology follows a simple protocol, does not use exotic reagents, is inexpensive and platform-independent, offers same-day results, and is versatile toward many sample types and applications. Scientists may even label Stellaris FISH probes with a number of various dyes, to allow for multiplex detection of different RNA targets simultaneously. Finally, scientists can address the stochastic nature of gene expression and visualize RNA through direct detection without isolation, purification, and amplification.
The Stellaris RNA FISH Method
The Stellaris 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.
Step 1: Prepare Sample
The sample is adhered or sectioned onto a #1 coverglass and permeabilized with 70% EtOH. 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, and do not support confocal microscopy with Stellaris FISH probes.
Sensitivity & Specificity
The large number of probes in a Stellaris FISH assay ensures a high level of sensitivity and specificity. The inherent redundancies of this direct detection method minimizes the possibility of false negatives. Positive signal is only identified from the combined localized fluorescence of multiple probes. Off-target binding of probes generates weak and diffuse fluorescence, well below the threshold for specifically-targeted mRNA detection. Furthermore, Stellaris FISH probes can bind to partially degraded target mRNAs making them well-suited for formalin-fixed, paraffin-embedded (FFPE) tissue samples.
Stellaris 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 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 FISH can also be combined with existing technologies such as qPCR, DNA FISH, IHC (immunohistochemistry), and western blotting to provide complementary information.
- Download a full version white paper which contains more details about Stellaris RNA FISH.
- Read our Application Note which provides details about positive controls in a Stellaris assay, multiplexing, and quantification of RNA in tissue samples.
- Use the Stellaris Getting Started Guide to plan and seamlessly perform Stellaris experiments.
- Visit our protocols page to learn more about the Stellaris FISH technique.
- If your target of interest in not available as a ShipReady or DesignReady Assay, try designing your own custom Stellaris probes by using our online Stellaris probe designer, free with registration.
- Order Stellaris Buffers for its ability to enhance signal and reduce background.
- Watch our full-length Stellaris promotional video that discusses the mechanism behind Stellaris FISH and its potential applications.
- BiteSize Bio Article: RNA Detection with Stellaris™ Fluorescence in situ hybridization (FISH) probes
- BiteSize Bio Article: It’s 10 am. Do you know where your mRNAs are?
Go to our Stellaris RNA FISH product page to find ordering information for Stellaris Buffers and Custom, DesignReady, and ShipReady Assays.
Stellaris 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.