What Is ACD Bio’s RNAscope Technology?

The study of gene expression, which dictates how genetic information is used to create functional products like proteins, is fundamental to biology. Advanced Cell Diagnostics (ACD), a Bio-Techne brand, developed RNAscope, a specialized in situ hybridization (ISH) technique designed to detect single RNA molecules directly within cells and tissues. This method allows researchers to see precisely where and to what extent specific genes are active.

Unlike methods that require grinding up tissue to extract RNA, which destroys spatial information, RNAscope preserves the sample’s morphological context. This preservation allows scientists and clinicians to connect gene activity to specific cell types or disease states.

The Science Behind RNAscope Technology

RNAscope technology is based on RNA in situ hybridization, where a labeled probe binds to a specific RNA sequence. What sets RNAscope apart is its unique probe design and signal amplification method. The probes, called double Z-probes or “ZZ” probes, are short strands of nucleic acids designed to bind in pairs to a target RNA molecule. Each “Z” probe has a lower region complementary to the target RNA and an upper region that acts as a binding site for the amplification structure.

For a signal to be generated, two “Z” probes must bind side-by-side on the target RNA sequence. This requirement for tandem hybridization is a reason for the technology’s high degree of specificity, as it is statistically improbable for two independent probes to bind next to each other on an off-target sequence. A set of about 20 such ZZ probe pairs are designed to bind along the length of a single target RNA molecule, ensuring a robust signal.

Once the probes are bound to the target RNA, signal amplification begins using a branched DNA or “tree” amplification system. A pre-amplifier molecule binds to the top sections of the paired Z-probes, creating a foundation. Multiple amplifier molecules then attach to the pre-amplifier, and subsequently, numerous labeled probes containing either a chromogenic enzyme or a fluorescent dye bind to the amplifiers. This sequential binding builds a structure that magnifies the signal, making it possible to detect a single molecule of RNA as a distinct dot under a microscope.

Diverse Applications of RNAscope

RNAscope technology is applied across a wide spectrum of research and clinical investigations. It can be used on various sample types, including formalin-fixed paraffin-embedded (FFPE) tissues, fresh-frozen samples, and cultured cells. The technology is used in many fields:

• Cancer research: To identify biomarkers for diagnosis and prognosis, study cellular diversity within tumors, and investigate mechanisms of drug resistance.
• Neuroscience: To map gene expression patterns across different brain regions and among various types of neurons to help unravel the complexity of neural circuits.
• Infectious disease research: For detecting viral RNA directly within infected tissues and studying the interactions between a host and a pathogen.
• Developmental biology: To track where and when genes are turned on and off during embryonic development.
• Drug discovery: To validate new drug targets and assess the efficacy and potential toxicity of therapeutic compounds.

Key Capabilities of the RNAscope Assay

A primary strength of the RNAscope assay is its specificity, derived from the double Z-probe design. This feature minimizes the risk of false positives from off-target binding.

The technology is also known for its sensitivity. The signal amplification system can make a single RNA molecule visible as a discrete dot. This capability is useful for detecting genes expressed at low levels and allows for precise quantification of gene expression on a cell-by-cell basis.

RNAscope provides spatial resolution, allowing researchers to see gene expression within the native tissue architecture. This contextual information is important for understanding a gene’s biological role by revealing which specific cell types are expressing it.

Some RNAscope assays offer multiplexing capabilities, enabling the simultaneous detection of multiple RNA targets within the same sample. Using different fluorescent labels for each target, researchers can study how different genes are expressed in relation to one another. This is useful for investigating gene co-expression patterns and understanding complex cellular pathways.

Visualizing and Interpreting RNAscope Results

The output of an RNAscope experiment is visualized using a microscope, where results appear as distinct, punctate dots within cells. Each dot represents a single molecule of the target RNA, providing a clear representation of gene expression at the single-cell level.

With chromogenic labels, these dots appear as colored spots visible with a bright-field microscope. In multiplex assays, different fluorescent dyes are used, and the dots appear in various colors when viewed with a fluorescence microscope. This allows researchers to distinguish between several different RNA targets in the same field of view.

Image analysis involves identifying the cell types containing the signals by correlating the data with tissue morphology. Researchers then assess the level of gene expression by counting the dots per cell, which can be done manually or with automated image analysis software. Observing the spatial distribution of these dots can also reveal gene expression patterns across a tissue section, providing insights into cellular function and organization.