When it comes to protein detection and characterization, antibodies are one of the most widely used and trusted reagents. For many decades, scientist have relied on antibodies to perform Western blotting, immunostaining and immunoprecipitations to monitor protein size, localization and even cellular interactions.
In recent years, there has been a lot of attention in regards to the specificity, reproducibility, and validation of antibodies, as this has been the source of many issues for researchers. Antibodies can cross-react and bind to more than one protein, or to the entirely wrong target, while still generating a staining pattern that may appear to detect a protein of the correct size or in the expected location. They can also be unexpectedly impeded by physical changes in the target protein folding that hides the antigen. Batch-to-batch variation in antibodies can waste time, money, and samples and completely derail a project. And in some cases, an antibody is not even available for the specific target you are measuring.
Because of these issues, scientists need an alternative assay that can be used instead of or in tandem with antibody-based assays to confirm their results. RNA fluorescence in situ hybridization, or FISH, is a solution for researchers that avoids the use of antibodies.
RNA FISH uses fluorescent oligonucleotide probes to bind to target RNA sequences. Direct in situ RNA FISH can be used to measure RNA transcript numbers and assess gene expression without any of the issues of antibody reproducibility, specificity, and availability.
RNA in situ hybridization is not a new technique, but recent advances are bringing the technology to a new level. Stellaris RNA FISH is an improved RNA FISH technology from LGC Biosearch that enables simultaneous detection, localization, and quantification of individual RNA molecules at the cellular level.
Stellaris RNA FISH uses a set of fluorescent RNA probes designed to bind along the length of the target RNA. The technology depends on the binding of at least 25 unique probes together on the target to register a visible fluorescence signal. Fluorescence of stray probes falls below the detection threshold and minimizes false positive results. You can see here, for example, that the punctate spots indicating positive signal clearly stand out from the background.
Stellaris probes are only about 20 nucleotides in length, meaning they can penetrate into tissue samples more effectively than longer probes or larger molecules like antibodies.
RNA FISH can be used on its own or in combination with immunodetection for validation and more thorough analysis. Stellaris is designed to be performed in the same assay as antibody staining, meaning you can simultaneously image a messenger RNA and the protein for which it codes.
Combining RNA FISH and immunodetection both validates your results and gives you deeper insights into gene expression. For example, you can identify mechanisms of translational regulation by imaging both mRNA and protein levels of the same gene.
Single-molecule RNA FISH combined with immunostaining can be used for a wide range of applications. Neuroscience researchers are studying disease states or region-specific expression in cells, tissues, or the entire intact brain. The expression profiles of various cancer states down to specific intracellular localization of RNA molecules can be examined with this methodology. You can target long non-coding RNA for epigenetic research; or study expression patterns in embryonic versus adult stem cells. You can even study pathogens like viruses and fungi within host cells.
All in all, RNA FISH is a fantastic tool for studying gene expression, both on its own and in concert with immunostaining. New advances in the technology are making it even more versatile and accessible to researchers.