Stellaris RNA FISH |
Stellaris™ RNA FISH: Detection, Localization, and Quantification of mRNA at the Cellular Level
What is Stellaris RNA FISH?Stellaris FISH (fluorescence in situ hybridization) is a RNA visualization method that allows simultaneous detection, localization, and quantification of individual mRNA 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 simultaneous 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
- Sensitivity & Specificity
- Multiplex Capability
- Additional Resources
- Order Information
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 buffer with short incubation periods is 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.
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.
Multiplex Capability
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.
Stellaris FISH can also be combined with existing technologies such as qPCR, DNA FISH, IHC (immunohistochemistry), and western blotting to provide complementary information.
Additional Resources
- 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.
- Visit our protocols page to learn more about the Stellaris FISH technique.
- Try designing your own custom Stellaris probes by using our online Stellaris probe designer, free with registration.
- Watch our full-length Stellaris promotional video that discusses the mechanism behind Stellaris FISH and its potential applications.
Order Information
Go to our Stellaris RNA FISH product page to find ordering information. Custom Stellaris FISH Probes costs $575 per probe set for 5 nmol of total pooled oligos and catalogued Stellaris FISH Probes costs $199 per probe set for 1 nmol of total pooled oligos.
This product is sold under license from the University of Medicine and Dentistry of New Jersey and may be used under its patent rights for Research Use Only.
