A New Tool for Visualizing RNA Transcripts in Brain Tissue Sections

Exploring gene expression of particular neuronal cell types can be quite difficult to identify in complex tissue samples. Current methodologies such as PCR or sequencing-based methods of gene expression are destructive in the sense that the tissue sample is consumed during the assay preventing subsequent manipulations. These have the secondary challenge of isolating precise populations of cells prior to analysis. Typically, researchers will employ microdissection of particular structures to capture a sample. But, this can introduce variability between samples due to the difficulty in dissecting the exact, same amount of expressing and non-expressing cells. Other approaches might enrich a particular cell population with methods such as immunopanning or cell sorting, which also may not capture an entirely pure cell population. In both of these expression quantification methods, you are required to disassemble the tissue to achieve an answer or data point. However, there is an alternate method to investigate RNA expression without disrupting tissue morphology using RNA fluorescence in situ hybridization (FISH). LGC Biosearch Technologies offers a simple method for RNA FISH with Stellaris® RNA FISH probes that enable simultaneous localization and quantification of cellular expression patterns. In the context of cultured cells, these probes can also provide single-molecule RNA expression patterns with high signal-to-noise ratio in an easy-to-use protocol. To demonstrate the utility of RNA FISH, we will discuss the application of Stellaris RNA FISH in neuroscience research.

Stellaris RNA FISH overview

In situ hybridization is a powerful technique for detecting nucleic acid targets within cells or tissue. Previous iterations of FISH techniques required time and effort to precisely optimize the experiment to achieve specificity and a robust signal. Therefore, the past limitation of RNA FISH was low sensitivity, complex procedures, and a limited ability to detect multiple targets. The Stellaris RNA FISH detection method allows simultaneous detection, localization, and quantification of individual RNA molecules in fixed tissue samples using fluorescence microscopy. A set of Stellaris probes consists of multiple, fluorescently-labeled oligonucleotides tiled against the complementary target sequence. When the entire probe set is hybridized to the intracellular RNA population and imaged, the result is sub-cellular resolution of individual molecules of RNA with a high signal-to-noise ratio. This large number of probes leads to a higher number of fluorophores attached to the target, which increases the signal. A typical Stellaris probe set contains a mixture of up to 48 of these target specific probes pooled together.

The Stellaris technology addresses many issues of gene expression through direct detection without the need for RNA isolation, purification, or amplification. The protocol is simple, easy to follow, and uses common reagents. Stellaris RNA FISH is highly versatile toward many sample types and applications, which can be seen in the application of this method in neuroscience research. In the past, neuronal tissue was recognized as a difficult sample type for in situ RNA detection. This was partially due to abundant lipid content and high complexity of the specialized structures within the brain. The use of Stellaris RNA FISH has overcome these barriers and enabled neuroscientists to easily localize multiple genes of interest down to the individual molecules of RNA. One example shown below is the use of a Stellaris probe set targeted to Olig2, a lineage marker of mature oligodendrocytes.  The fluorescent signal from each mRNA appears is displayed in pseudocolor as white puncta within the tissue with DAPI staining in blue.  

Neuroscience application

The ability to isolate precise cellular populations in the brain becomes a serious challenge due to the composition of brain tissue. The brain is made of not only neurons, but also a wide variety of cell types with unique characteristics that can pose a challenge for RNA detection. Furthermore, the brain is regionally heterogeneous containing specialized structures, where each region could perform distinct and unique functions, which can affect gene expression. Finally, there is additional specialization on a per cell basis within these structures and regions, which pose challenges for non-RNA FISH methods.

Stellaris overcomes challenges in working with brain tissue

The following table below highlights various challenges in working with neuronal tissue and how the Stellaris method overcomes these challenges.

Stellaris RNA FISH is a straightforward, affordable, and sensitive FISH method that is opening new avenues of research in neuroscience. Want to learn more? Download the Stellaris Getting Started Guide for more background information and read the full set of guidelines for using Stellaris RNA FISH in brain sections.

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