What Are RNAscope Probes and How Do They Work?
Discover how scientists pinpoint gene activity with single-molecule precision, revealing which cells are active and where they are located within tissues.
RNAscope probes are tools in molecular biology that allow scientists to see specific RNA molecules directly within cells and tissues. This technology provides a visual map of gene activity by showing exactly where certain genes are being turned on, revealing its impact on biological and medical research.
The Core Principle of RNAscope Probes
RNAscope technology uses a unique probe design and signal amplification system. The core of this technology is the “double Z” target probe, which is shaped like the letter Z. Each probe has two distinct parts: a lower region that binds to the target RNA sequence and an upper region that serves as a docking site for amplification machinery.
For a signal to be generated, two separate “Z” probes must bind to the target RNA right next to each other. This adjacent binding creates a complete docking site on the upper portions of the probes for a signal pre-amplifier molecule to attach. This requirement for two independent binding events is a security check that reduces the chance of a false positive signal.
Once the probes are correctly bound, a signal amplification cascade begins. Multiple amplifier molecules bind to each pre-amplifier, and these amplifiers, in turn, bind numerous labeled probes carrying a fluorescent molecule or an enzyme. This branching amplification structure creates a strong signal from just a single RNA molecule, making it visible under a microscope. An assay often uses a pool of about 20 probe pairs for one target, ensuring robust detection.
Detecting RNA Within Its Native Environment
A defining feature of RNAscope technology is its ability to detect RNA “in situ,” meaning in its original place. This allows researchers to visualize gene expression within the preserved architecture of a tissue sample. Scientists can see which specific cells are expressing a particular gene and how those cells are organized. This spatial context is important for understanding the interplay between different cell types.
When a scientist examines a sample treated with RNAscope probes, they see distinct dots. Each dot represents a single molecule of the target RNA, appearing directly within the cellular structures. Depending on the assay, these signals can be fluorescent, appearing as bright, colored dots, or chromogenic, showing up as colored precipitates visible with a brightfield microscope.
This visualization provides direct evidence of gene activity at a specific location. For instance, a researcher can determine if a gene is active in nerve cells but not adjacent support cells. This level of detail is lost in techniques that measure average RNA levels from homogenized tissue.
Distinctive Capabilities of RNAscope
The design of RNAscope probes provides several capabilities. Its high sensitivity is a direct result of the branched signal amplification system, allowing for the reliable detection of even a single RNA molecule. This is useful for studying genes expressed at very low levels. The double-Z probe design ensures high specificity by minimizing off-target signals and allowing distinction between closely related RNA sequences.
Another feature is multiplexing, the simultaneous detection of multiple RNA targets in the same tissue sample. Using different fluorescent colors for each target, researchers can visualize the co-expression of several genes at once. Advanced assays can detect up to 12 different targets, providing a detailed snapshot of how multiple genes are interacting within the same cell.
The technology is also versatile. It can be applied to a wide variety of sample types, including formalin-fixed paraffin-embedded (FFPE) tissues, fresh-frozen samples, and cultured cells. This adaptability allows work with clinically relevant archival tissues. Probes can also be designed to detect different kinds of RNA, including messenger RNA (mRNA) and non-coding RNAs.
RNAscope in Action: Research and Discovery
RNAscope technology has applications across biological and medical research. In cancer research, it is used to characterize tumor heterogeneity. Scientists can visualize different cell types within a tumor—such as cancer cells and immune cells—and study their communication by examining the expression of signaling molecules. This can help identify biomarkers for diagnosis or to predict a patient’s response to therapy.
In neuroscience, RNAscope is used to map gene activity across different brain regions with single-cell resolution. Researchers can visualize the expression of neurotransmitter receptors to understand how neural circuits function or how they are altered in neurological disorders, such as Alzheimer’s disease or psychiatric disorders.
The technology is also instrumental in infectious disease research. Scientists use it to locate viral RNA, such as from influenza or SARS-CoV-2, directly within infected tissues to identify which cell types are targeted. In drug development, RNAscope serves as a tool to verify the action of new therapeutics by confirming a drug is turning genes on or off in the correct target cells.