Hybrid Nucleic Acid Detection Signal Amplification in FFPE Tissue
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Solution Overview
Problem
Current in situ hybridization (ISH) techniques, including RNAscope®, face challenges in detecting low copy number DNA or RNA targets in Formalin-Fixed, Paraffin-Embedded (FFPE) tissue sections due to RNA degradation and limited sensitivity and specificity, especially at 40X magnification.
Innovation Solution
Combining RNAscope® with biotin-(strept)avidin, antibody, and Tyramide Signal Amplification (TSA) methods to create a hybrid signal amplification system that enhances sensitivity and specificity, achieving robust signal amplification with improved signal-to-noise ratio and consistency for nucleic acid detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNAscope® is used for nucleic acid detection in FFPE tissue sections, then sensitivity is improved, but detection reliability deteriorates due to RNA degradation and low signal-to-noise ratio
Solution Approach 1:
The patent combines RNAscope® technology with Tyramide Signal Amplification (TSA) to create a hybrid detection system. The RNAscope® capture probes specifically bind to target nucleic acids while TSA provides robust signal amplification through horseradish peroxidase-catalyzed deposition of tyramide-conjugated haptens. This merging of two complementary technologies resolves the contradiction by maintaining the specificity of RNAscope® while adding the amplification power of TSA to improve reliability in degraded FFPE samples.
Solution Approach 2:
The patent modifies the signal amplification parameters by introducing multiple amplification steps (capture probe hybridization followed by TSA amplification). The TSA step uses enzymatic catalysis to deposit numerous hapten molecules at each binding site, effectively changing the signal intensity parameter. This parameter change enables reliable detection even when RNA is partially degraded, as the amplified signal overcomes the low signal-to-noise ratio problem.
2Measurement precision
If signal amplification is increased to detect low copy number targets, then detection sensitivity is improved, but background noise increases
Solution Approach 1:
The patent uses capture probes as intermediaries that specifically bind to target nucleic acids before TSA amplification occurs. These capture probes act as selective mediators that ensure only target-bound sites undergo amplification. The TSA reagents (horseradish peroxidase and tyramide) are introduced as controlled intermediaries that amplify signal only at locations where capture probes have successfully hybridized to targets, thereby minimizing background noise while maximizing sensitivity for low copy number detection.
3Stability of the object's composition
If formalin fixation and paraffin embedding is used to preserve tissue morphology, then morphological preservation is improved, but nucleic acid accessibility deteriorates due to cross-linking
Solution Approach 1:
The patent applies preliminary tissue processing steps including deparaffinization and antigen retrieval before hybridization. These preliminary actions reverse the cross-linking effects of formalin fixation and paraffin embedding, restoring nucleic acid accessibility. The deparaffinization step removes paraffin wax that blocks probe access, while antigen retrieval techniques (such as heat-induced epitope retrieval) break formalin cross-links. These preliminary actions enable subsequent capture probe hybridization to occur efficiently on FFPE tissue while preserving the morphological context.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The combined method enables reliable detection of nucleic acids with high intensity and low background noise, allowing for easy visualization at 10X magnification and consistent results suitable for routine diagnostic pathology.
Implementation Method 1
The capture probes hybridize to both target nucleic acid and signal generating multimer and thus capture signal generating multimer to target nucleic acid
Implementation Method 2
The label probe is capable of binding to a label particle or molecule that provide detectable signal
Implementation Method 3
RNAscope® in combination with biotin-(strept)avidin
Implementation Method 4
RNAscope® in combination with an antibody
Implementation Method 5
RNAscope® in combination with Tyramide Signal Amplification (TSA)
Data Source
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AI summary
The invention provides a method of detecting at least one target nucleic acid in a cell by in situ hybridization, a fixed and permeabilized cell, a tissue section comprising a cell, and a kit for detecting at least one target nucleic acid in a cell.