RNA In Situ Hybridization (ISH) is a method used to precisely locate and visualize specific RNA molecules directly within a cell or tissue section. This technique identifies which cells are actively expressing a particular gene using a labeled probe complementary to the target RNA sequence. By maintaining the native spatial arrangement of cells, RNA ISH provides a unique view of gene activity, unlike methods that measure expression from homogenized samples. The core principle relies on nucleic acids’ thermodynamic property, where complementary single strands anneal, or hybridize, to form a stable double-stranded structure. This approach is fundamental to understanding cellular function, disease progression, and the complex architecture of gene expression.
Fundamentals of Sample Preparation and Hybridization
The process begins with careful preparation of the biological sample to ensure the target RNA molecules are preserved and remain accessible. Tissue fixation is the initial step, commonly achieved using agents like 4% paraformaldehyde or formalin, which create cross-links between molecules to lock cellular components in place. While fresh-frozen tissue can also be used, chemical fixation is often preferred for maintaining tissue morphology, particularly for archival samples like formalin-fixed, paraffin-embedded (FFPE) blocks. The tissue is then cut into very thin sections, often three to seven micrometers thick, before being mounted onto slides.
Following sectioning, the cell membranes and cytoplasm must be made permeable to allow probe molecules to enter the cell and reach their targets. Permeabilization is achieved through treatments with detergents, such as Triton X-100, or organic solvents, like ethanol, which create microscopic pores in the lipid membranes. Protease use, such as Proteinase K, digests proteins that may shield RNA transcripts, unmasking the target sequences and improving the hybridization signal. However, this digestion must be carefully controlled, as excessive treatment can compromise the cell’s structure or lead to the loss of the RNA itself.
The hybridization phase involves incubating the prepared tissue with the complementary nucleic acid probe under specific, controlled conditions. This step is governed by stringency, which is the balance of temperature, salt concentration, and formamide levels that dictate the specificity of the binding. A higher stringency environment encourages only perfectly matched probe-target hybrids to remain stable, while weakly bound, non-specific probes detach. Following hybridization, a series of washing steps moves from higher to lower salt concentrations and adjusts the temperature to remove any remaining unbound probes. This washing is essential to reduce background noise and ensure that only the signal from specifically bound probes is detected.
Principles of RNA Probe Design and Chemistry
The specificity and sensitivity of RNA ISH are largely determined by the design and chemical composition of the nucleic acid probes. Historically, longer riboprobes—single-stranded RNA molecules labeled with a hapten—were used, but modern techniques often rely on pools of shorter, synthetic oligonucleotide probes. A typical probe set for a single target RNA sequence may consist of 20 to 50 oligonucleotides designed to bind along the length of the target transcript. This approach ensures that even if one part of the target RNA is partially degraded or inaccessible, the other probes in the pool can still bind.
Each oligonucleotide probe is chemically modified to allow for subsequent detection, often by incorporating a label such as a fluorophore for direct visualization or a hapten like digoxigenin or biotin. Haptens are small molecules that do not produce a signal themselves but are recognized by labeled antibodies or proteins later in the protocol. Modern probe architectures, such as the patented “double Z” design used in the RNAscope system, are engineered with a two-part structure. The lower region is complementary to the target RNA, while the upper region acts as a binding site for the signal amplification cascade.
A key advancement in probe design is the incorporation of signal branching mechanisms, which are intrinsic to the probe architecture. In the double Z system, two adjacent Z-probes must bind in tandem to the target RNA to create a functional binding site for a pre-amplifier molecule. This requirement ensures that non-specific, single-probe binding events do not lead to signal amplification, suppressing background noise and increasing specificity. Other techniques, such as the branched DNA (bDNA) assay, use the bound probe as a scaffold upon which a cascade of complementary DNA molecules can hybridize, creating a structure that increases the number of available binding sites for the final label.
Signal Amplification and Visualization Techniques
Once the probe has hybridized to the target RNA, signal amplification is often necessary, especially for transcripts present at low copy numbers, to make the signal detectable. The amplification cascade used in many commercial assays involves sequential hybridization steps. A pre-amplifier binds to the probe, followed by multiple amplifier molecules binding to the pre-amplifier, and finally, many labeled reporter probes attaching to the amplifiers. This layered approach can result in the accumulation of hundreds or thousands of reporter molecules at the site of a single RNA target, enabling single-molecule detection capability.
Another enzymatic amplification method is Tyramide Signal Amplification (TSA), sometimes referred to as Catalyzed Reporter Deposition. In this technique, the hybridized probe is ultimately detected by an enzyme, typically Horseradish Peroxidase (HRP), which is conjugated to an antibody or streptavidin. The HRP then uses hydrogen peroxide to activate a labeled tyramide molecule, causing it to covalently bind to tyrosine residues on proteins in the immediate vicinity of the target. This localized deposition of numerous labeled tyramide molecules can increase the fluorescent signal intensity by up to 100-fold, making it suitable for detecting transcripts with very low abundance.
The final detection method determines the visualization technique, with the two forms being Fluorescent In Situ Hybridization (FISH) and Chromogenic In Situ Hybridization (CISH). FISH utilizes probes labeled with fluorophores that emit light when excited by a specific wavelength, allowing visualization using a fluorescence microscope. The advantage of FISH is the ability to multiplex, or simultaneously detect multiple RNA targets, by using distinct fluorophores for each probe set. CISH uses an enzyme-based reaction, often involving HRP, to convert a soluble substrate into a colored, insoluble precipitate at the target site. This precipitate is visible with a standard bright-field microscope, offering a stable, non-fading signal that can be stored long-term and viewed concurrently with the tissue’s morphology.
Quantitative Analysis and Data Interpretation
Quantitative analysis of RNA ISH data provides measurable metrics of gene expression at the single-cell level. This process involves image analysis software designed to automatically detect and count the discrete signal dots generated by the amplification systems. For single-molecule techniques, each detected dot often represents an individual RNA transcript, allowing for the quantification of transcript copy number per cell. Algorithms are employed to segment the image, distinguishing individual cells and their subcellular compartments, such as the nucleus and cytoplasm, to determine the precise localization of the RNA signals.
Quantification establishes thresholds for positive and negative cells, enabling researchers to define populations based on their expression levels of a particular RNA target. The software calculates metrics like the average number of dots per cell, the percentage of positive cells within a specific tissue region, and the spatial distribution of the signals. For instance, a researcher can quantify the difference in gene expression between immune cells and tumor cells within a heterogeneous tissue sample, providing cell-type-specific expression data that preserves the tissue architecture.
The spatial context is an important element of data interpretation, as the location of the RNA signal within the cell or tissue can provide functional insights. Observing transcripts clustered near the nucleus may suggest a high rate of transcription, while cytoplasmic localization indicates translation readiness. The ability to simultaneously quantify multiple RNA targets (multiplexing) allows for the calculation of co-expression patterns, revealing which genes are active within the same cell and how different cell types interact with their neighbors. This data transforms the visualization into statistically relevant information, supporting conclusions about gene regulation and cellular heterogeneity.