Seeing RNA in Context

The Challenge of Seeing Single RNA Molecules

Why has visualizing RNA in tissues been so challenging? The answer lies in both the delicate nature of RNA molecules and their relatively low abundance within cells. Unlike DNA, which exists in two copies per cell, RNA molecules are single-stranded and easily degraded by enzymes called RNases that seem to be everywhere in our environment. Additionally, while some RNA species are abundant, many messenger RNAs (mRNAs) that code for proteins exist in only a few copies per cell, making them exceptionally difficult to detect against background noise.

Visualizing RNA within intact tissues requires advanced technologies that preserve spatial context while providing sufficient sensitivity.

Traditional methods like immunohistochemistry detect proteins rather than RNA, while conventional in situ hybridization techniques often lack the sensitivity to detect low-abundance RNAs or struggle with multiplexing—the simultaneous detection of multiple targets. What makes ViewRNA technology particularly powerful is its ability to overcome both these limitations through an elegant signal amplification strategy that doesn't amplify background noise, providing exceptional signal-to-noise ratio while detecting multiple RNA targets in their precise cellular locations ^{1 2} .

A Step-by-Step Look at the ViewRNA Workflow

Tissue Preparation: Setting the Stage

The ViewRNA process begins with careful tissue preparation to preserve both RNA integrity and tissue morphology. For formalin-fixed paraffin-embedded (FFPE) tissues—the standard preservation method in pathology labs worldwide—this involves baking slides at 60°C to promote adhesion, followed by deparaffinization in xylene and rehydration through a series of ethanol washes. A critical heat pretreatment step at 90-95°C then exposes the target RNA molecules by breaking protein cross-links, making the RNA accessible for probing ⁵ .

For cryopreserved tissues, the process begins with fixation in formaldehyde to maintain cellular structures. Both sample types then undergo protease digestion—a carefully optimized treatment with enzymes that gently digests proteins obscuring the RNA targets without damaging the RNA itself. This delicate balance is crucial: too little digestion and the probes can't reach their targets; too much and both tissue structure and RNA may be compromised ⁵ .

The Hybridization Cascade: Precision and Amplification

Once the tissue is prepared, the core branched DNA detection process begins. This represents a carefully choreographed molecular dance with three main stages ^{1 5} :

Between each hybridization step, rigorous washing procedures remove any unbound molecules, ensuring that only specifically bound components remain and minimizing background signal. The entire process occurs in a carefully controlled humidified chamber at 40°C to promote specific hybridization while preventing evaporation that could damage the tissue samples ⁵ .

Visualization and Analysis: Seeing the Unseen

For visualization, samples are typically counterstained with DAPI—a blue fluorescent dye that binds to DNA in the cell nucleus, providing essential architectural context by highlighting individual cells. The slides are then imaged using a fluorescence microscope equipped with specific filter sets matched to the fluorophores used in the experiment ¹ .

Multiplex fluorescence imaging reveals distinct RNA localization patterns within tissue architecture.

The result is a stunning multicolor image where individual RNA molecules appear as distinct dots within cells, allowing researchers not only to identify which cell types express specific genes but also to count RNA molecules and analyze their subcellular localization—whether concentrated in certain cellular compartments or distributed throughout the cytoplasm. This level of detail enables unprecedented insights into gene regulation at the single-cell level within intact tissues ² .

Transforming Biomedical Research Through Spatial Context

The ability to visualize multiple RNA molecules simultaneously within their native tissue environment is driving discoveries across numerous fields of biology. In cancer research, scientists can now identify distinct cell populations within tumors—cancer cells, immune cells, and stromal cells—based on their gene expression patterns, revealing how cellular interactions influence disease progression and treatment response. In neuroscience, researchers can map neurotransmitter receptors and synaptic proteins across different brain regions, uncovering the molecular basis of neural circuits ² .

Spatial transcriptomics enables researchers to study gene expression within the context of tissue architecture.

Multiplex imaging reveals complex cellular relationships that are invisible to bulk analysis methods.

The technology also enables direct detection of splice variants, non-coding RNAs, and viral RNAs, providing insights into gene regulation during development and disease. Unlike bulk sequencing methods that average expression across all cells, the ViewRNA approach preserves cellular heterogeneity, allowing researchers to identify rare cell populations that might play disproportionate roles in biological processes ² .