Technology

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.

Applicability

The Stellaris RNA FISH method is applicable to a variety of biological specimens, including bacteria, yeast, mammalian cells, C. elegans embryos and L1-L2 larvae, Drosophila melanogaster wing imaginal discs, and primary rat hippocampal neurons. Samples with a thickness of less than 15 µm are most suitable for this method. Sample thickness is a concern, because light scatter and diffusion of emitted light increases with thickness. While it is possible that one could obviate this restriction through the use of confocal imaging, the lower sensitivity of confocal microscopy significantly hinders its applicability in this context.

Stellaris FISH is an effective tool for several key applications.  For example, Stellaris probes can be used to reveal up- and down- regulation of gene expression in drug screening studies.  Another example is the characterization of tumors by correlating mRNA levels of known oncogenes.  Stellaris FISH can also be combined with existing technologies such as qPCR, DNA FISH, IHC (immunohistochemistry), and western blotting to provide complementary information.

Design Considerations

Three major design considerations to target coding sequences of gene transcripts are 1) mRNA length, 2) repeat sequences and 3) sequences of low complexity (such as GC content). As for length, the number of probes that can be accommodated for robust detection is primarily sequence dependent. Although mRNAs as short as 550 nucleotides have been detected using only 20 probes, we recommend 48 probes whenever possible. Repetitive elements and low complexity sequences must be screened in advance and manually removed, further restricting the sequence space available for design. For this reason, certain genes such as the keratins may present unusual challenges. The optimal target has an overall GC content of 40-50%, although mRNAs with higher GC content may yield good results by using more stringent washing conditions.