Simultaneous RNA, DNA, and Protein Detection in FFPE Tissue
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Solution Overview
Problem
Current methods for detecting RNA in formalin-fixed paraffin-embedded tissue are often performed after extensive protease treatment, which is incompatible with downstream protein detection, limiting the ability to analyze RNA and protein targets simultaneously in the same sample.
Innovation Solution
A method involving in situ hybridization using labeled nucleic acid probes for RNA, followed by antigen retrieval and antibody-based detection for proteins, with optional protease treatment for DNA detection, allowing for simultaneous detection of RNA, DNA, and proteins in the same biological sample without protease treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If protease treatment is performed to access DNA targets, then DNA detection is enabled, but protein epitopes are degraded and protein detection becomes impossible
Solution Approach 1:
The detection process is segmented into separate sequential steps: first detecting RNA targets via in situ hybridization, then detecting protein targets via immunohistochemistry, and finally detecting DNA targets via in situ hybridization after protease treatment. This segmentation allows each detection type to be performed under optimal conditions without compromising the others.
Solution Approach 2:
RNA detection is performed as a preliminary action before protease treatment. This ensures that RNA targets are detected while still intact, before the protease degrades proteins. The preliminary RNA detection step captures information that would otherwise be lost or compromised by subsequent protease treatment.
2Adaptability or versatility
If RNA detection is performed after protease treatment, then DNA targets are accessible, but RNA targets are degraded and RNA detection fails
Solution Approach 1:
RNA detection is performed as a preliminary action before any protease treatment. This timing ensures that RNA targets are detected while still intact and accessible, before the protease begins degrading proteins and potentially affecting RNA stability. The method captures RNA information at the optimal time point.
3Productivity
If multiple targets are detected simultaneously, then comprehensive analysis is achieved, but sample preparation requirements conflict and detection accuracy decreases
Solution Approach 1:
The simultaneous detection of multiple targets is achieved through temporal segmentation of the detection process. Different target types (RNA, protein, DNA) are detected in sequential steps, each optimized for its specific requirements. This segmented approach maintains high detection accuracy for each target type while still achieving comprehensive multi-target analysis from a single sample.
Solution Approach 2:
The detection process uses periodic action by cycling through different detection modalities: in situ hybridization for RNA, immunohistochemistry for proteins, and in situ hybridization for DNA. Each modality is applied periodically in sequence, allowing optimal detection conditions for each target type to be maintained while achieving comprehensive analysis.
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
Enables better correlation between RNA, DNA, and protein expression, improving disease analysis by enabling simultaneous detection of multiple targets in a single sample without compromising sample integrity.
Implementation Method 1
subjecting the sample to an in situ hybridization reaction using a labeled nucleic acid probe that directly or indirectly binds an RNA target
Implementation Method 2
subjecting the sample to an in situ hybridization reaction using an antibody-based method and attaching one or more antibody probe to antigens on the sample
Data Source
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AI summary
Novel methods of probing multiple targets in a biological sample are provide whereby the targets are DNA, RNA and protein. The method comprises subjecting the sample to an in situ hybridization reaction using a labeled nucleic acid probe that binds an RNA target, observing a signal, and optionally removing the signal. The method further comprises an antigen retrieval protocol, observing a signal, removing the signal, and optionally applying a protease treatment to access the sample's DNA targets by subjecting the sample to an in situ hybridization reaction using a labeled nucleic acid probe, observing a signal from the labeled DNA targets, and optionally removing the signal.