RNAscope and HCR (Hybridization Chain Reaction) are two different methods of performing in situ hybridization, a technique used to visualize and localize specific RNA molecules within cells or tissues. While both methods are based on the principles of hybridization between RNA probes and target RNA molecules, they differ in their underlying mechanisms and applications.
The choice between HCR and RNAscope depends on factors such as probe design flexibility, experimental optimization requirements, signal amplification needs, cost considerations, and the specific goals of your study. It is recommended to evaluate these factors and consider the strengths and limitations of each method before making a decision. Below are some factors to consider.
RNAscope:
RNAscope is a proprietary technique developed by Advanced Cell Diagnostics (ACD) that allows for highly sensitive and specific detection of RNA transcripts at the single-molecule level. It employs a unique probe design strategy and signal amplification system.
Key features of RNAscope:
- Probe Design: RNAscope probes are designed as short oligonucleotides (typically 20-25 bases) that target specific RNA sequences of interest. Each probe is labeled with multiple adjacent oligonucleotide "Z" sequences, forming a "Z-probe." These Z-probes hybridize to the target RNA, forming a target-specific "Z-probe/target RNA" complex. https://acdbio.com/all-about-probes
- Signal Amplification: RNAscope uses a signal amplification method called "Branched DNA" (bDNA) amplification. Multiple pre-amplifier and amplifier molecules, each labeled with specific oligonucleotide sequences, are sequentially hybridized to the Z-probe/target RNA complex. This results in the amplification of the signal, enhancing the sensitivity of detection.
- Visualization: The amplified signal is detected using fluorescently labeled probes that specifically bind to the amplification products. This allows for visualization and localization of the target RNA molecules within cells or tissues using fluorescence microscopy.
Applications: RNAscope is widely used in research and clinical applications to study gene expression, RNA localization, and cellular heterogeneity. It can be applied to various sample types, including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues, and cell cultures.
HCR (Hybridization Chain Reaction):
HCR is another in situ hybridization technique that enables the visualization of RNA transcripts in cells or tissues. It relies on a chain reaction of hybridization events to amplify the signal.
Key characteristics of HCR:
- Probe Design: HCR probes are designed as two separate sets of DNA hairpin probes, called "initiator" and "amplifier" probes. The initiator probe is designed to hybridize to the target RNA molecule and open up, exposing a target-specific sequence. The amplifier probes, which have complementary sequences to the exposed sequence on the initiator probe, then hybridize and form a chain of amplifiers.
- Signal Amplification: The hybridization chain reaction occurs when multiple amplifier probes bind to a single initiator probe, leading to the formation of long amplification polymers. These polymers serve as a scaffold for the amplification process, resulting in the accumulation of amplifiers on the target RNA molecule.
- Visualization: The accumulated amplifiers can be labeled with fluorophores or other detection molecules to allow for visualization using fluorescence microscopy. The amplifiers bind to the target
HCR PROS:
The choice between HCR in situ hybridization and RNAscope depends on several factors, including the specific research goals, experimental requirements, and sample characteristics. Here are a few reasons why you might choose HCR in situ hybridization over RNAscope:
- Signal Amplification: HCR utilizes a chain reaction of hybridization events, which can lead to signal amplification. While RNAscope also employs amplification strategies, HCR offers the potential for longer amplification chains, resulting in increased signal amplification. This can be particularly beneficial for visualizing low-abundance or sparse RNA transcripts.
- Cost Considerations: Depending on the specific requirements and resources available, the cost of reagents and probes may be a factor in the decision. HCR can potentially be less expensive compared to commercially available RNAscope probes, especially when custom probe design is required.
HCR CONS:
HCR in situ hybridization has certain limitations that should be considered.
Here are some of the limitations associated with HCR:
- Background Signal: HCR can sometimes produce background signal, which refers to nonspecific staining or fluorescence that is not related to the target RNA of interest. This background signal can arise from nonspecific hybridization of the HCR probes or amplifiers to off-target RNA molecules. Careful optimization of the experimental conditions, including probe design and hybridization conditions, can help minimize background signal.
- Sensitivity: While HCR can provide signal amplification, its sensitivity may not be as high as some other methods, such as RNAscope or other amplification-based techniques. This can limit the detection of low-abundance RNA transcripts or transcripts that are expressed in a sparse manner within cells or tissues.
- Probe Design Complexity: The design and synthesis of HCR probes and amplifiers can be more complex compared to other in situ hybridization methods. HCR probes require the design of initiator and amplifier probes with complementary sequences, which adds an additional level of complexity and may require careful optimization to ensure specificity and efficiency. Molecular Instruments has streamlined the process to make it easier for you to pick your targets. https://www.molecularinstruments.com/hcr-rnafish
- Signal-to-Noise Ratio: Achieving a high signal-to-noise ratio is essential for accurate and reliable detection. However, variations in the amplification efficiency or specificity of HCR probes can affect the signal-to-noise ratio, leading to decreased sensitivity or specificity of the assay.
- Compatibility with Sample Types: HCR may have limitations when applied to certain sample types, such as formalin-fixed paraffin-embedded (FFPE) tissues. The fixation and processing methods used for FFPE samples can affect the accessibility of RNA molecules, leading to reduced efficiency of hybridization and signal amplification.
*** The performance and limitations of HCR can vary depending on the specific experimental conditions, probe design, and target RNA sequence. Optimizing the assay and carefully controlling the experimental variables can help overcome some of these limitations and improve the performance of HCR in situ hybridization. The NIF core will work with each sample to optimize the process and recommend RNAscope in those samples that do not meet the threshold for quality.
RNAscope PROS:
There are several reasons why you might choose to use RNAscope over HCR in situ hybridization for your specific experimental needs:
Sensitive and Specific Detection: RNAscope is known for its high sensitivity and specificity in RNA detection. The proprietary probe design strategy and signal amplification system of RNAscope enable the detection of individual RNA molecules with minimal background noise. This sensitivity is particularly beneficial when working with low-abundance or sparse RNA transcripts.
Multiplexing Capability: RNAscope allows for multiplexed RNA detection, meaning that it is possible to visualize and quantify multiple RNA targets simultaneously within the same sample. This can be achieved by using probes with different fluorophores or chromogenic labels, allowing for the examination of co-expression patterns or interactions between multiple RNA molecules.
Commercially Available Probes: RNAscope offers a wide range of commercially available probes targeting various RNA sequences, including those derived from the human, mouse, and rat genomes (or any other plant or animal). These pre-validated probes can save time and effort in probe design and optimization, especially when working with well-characterized genes or species.
Established and Validated Method: RNAscope has been extensively used and validated in numerous research studies and clinical applications. It has a strong track record of reliability and sensitivity for RNA detection in different sample types, including FFPE tissues. This established method may be advantageous if you prefer to use a well-established technique with proven performance.
Wide Range of Sample Types: RNAscope is compatible with various sample types, including FFPE tissues, frozen tissues, and cell cultures. This versatility allows for the analysis of RNA expression patterns in diverse experimental settings and sample collections.
RNAscope CONS:
RNAscope, as a widely used in situ hybridization technique, has several limitations that should be considered. Here are some of the limitations associated with RNAscope:
Probe Design Constraints: RNAscope requires the availability of specific probe sequences that target the RNA of interest. Designing high-quality and specific probes can be challenging, especially for certain RNA sequences with high homology or regions with complex secondary structures. In such cases, probe design may require careful optimization or alternative strategies.
Sensitivity to Target RNA Expression Levels: RNAscope may have limitations in detecting low-abundance RNA transcripts or transcripts expressed at a sparse level within cells or tissues. The sensitivity of RNAscope is influenced by the target RNA's expression levels, probe design, and the efficiency of signal amplification. As a result, detection of very low levels of RNA expression can be challenging.
Signal-to-Noise Ratio: Achieving a high signal-to-noise ratio is crucial for accurate detection and interpretation of results. However, variations in probe specificity, autofluorescence or nonspecific binding can contribute to background signal, leading to reduced signal-to-noise ratio.
Tissue Penetration and Accessibility: The ability of the RNAscope probes to penetrate and hybridize to target RNA molecules can be influenced by tissue characteristics, such as sample thickness, cellular density, and the presence of extracellular matrix components. These factors can impact the efficiency of probe penetration and hybridization, potentially limiting the detection of RNA within deeper regions of tissues. The core facility has protocols in place for thick tissue. Maximum penetration is approximately 80um.
Cross-Reactivity and Off-Target Effects: Although RNAscope probes are designed to be highly specific, there is still a possibility of cross-reactivity or off-target effects, especially when working with highly homologous RNA sequences or closely related gene families. Careful probe design, specificity validation, and appropriate controls are important to address these concerns.
It's important to note that RNAscope has been widely used and validated in many research and clinical applications. It offers a robust and established platform for sensitive and specific RNA detection, and it may be more suitable for certain applications or sample types, such as FFPE tissues.