Multiplex RNA In Situ Hybridization With Cleavable Label Detection
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Solution Overview
Problem
Existing RNA in situ hybridization (ISH) methods face limitations in multiplexing capability due to the small number of spectrally distinct fluorescent dyes, leading to substantial loss of nucleic acid detection sensitivity and cellular morphology, and are time and labor intensive.
Innovation Solution
A method involving multiple rounds of target probe hybridization, pre-amplification, and label detection using distinguishable cleavable labels to simultaneously detect multiple nucleic acid targets, allowing for higher multiplexing without repeated hybridization and detection steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If serial rounds of hybridization and detection are performed to increase multiplexing, then the number of detectable target sequences increases, but nucleic acid detection sensitivity and cellular morphology are substantially lost
Solution Approach 1:
The method segments the detection process into distinct phases: (1) simultaneous hybridization of multiple target probes to different nucleic acid targets in a single round, (2) pre-amplification of probe-target hybrids, and (3) sequential label detection with cleavable labels. This segmentation allows multiplexing while preserving sensitivity by avoiding repeated hybridization cycles that damage the sample.
Solution Approach 2:
The method performs preliminary pre-amplification of the target-probe hybrids before final label detection. This preliminary amplification step increases the signal strength early in the process, allowing subsequent label detection without requiring repeated hybridization rounds that would compromise sample integrity and detection sensitivity.
2Adaptability or versatility
If serial rounds of hybridization and detection are performed to increase multiplexing, then the number of detectable target sequences increases, but the process becomes time and labor intensive
Solution Approach 1:
The method segments the detection process into distinct phases: (1) simultaneous hybridization of multiple target probes to different nucleic acid targets in a single round, (2) pre-amplification of probe-target hybrids, and (3) sequential label detection with cleavable labels. This segmentation allows multiplexing while preserving sensitivity by avoiding repeated hybridization cycles that damage the sample.
Solution Approach 2:
The method maintains continuous useful action by performing all target probe hybridizations simultaneously in one step, followed by continuous pre-amplification and label detection processes. This eliminates the repeated hybridization-detection cycles of traditional methods, significantly reducing the total time required for multiplexed detection while maintaining high sensitivity.
3Adaptability or versatility
If more spectrally distinct fluorescent dyes are used to increase multiplexing, then the number of detectable targets increases, but the limitations of optical systems prevent further multiplication
Solution Approach 1:
The method introduces intermediary pre-amplifier molecules that bind to target-probe hybrids and provide multiple binding sites for label probes. This intermediary amplification system allows a single optical channel to detect multiple targets through signal amplification rather than requiring multiple spectrally distinct dyes, thereby increasing multiplexing capability without increasing optical system complexity.
Solution Approach 2:
The method changes the detection parameter from relying on spectral distinction of multiple fluorescent dyes to relying on signal amplification through pre-amplifier and label probe complexes. This parameter change allows detection of more targets within the same spectral range, effectively increasing multiplexing capability without requiring more complex optical systems with additional spectral channels.
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 simultaneous visualization of a higher number of target sequences with improved sensitivity and reduced time and labor, preserving cellular morphology.
Implementation Method 1
each target probe set comprises a pair of target probes that specifically hybridize to a target nucleic acid
Implementation Method 2
each pre-amplifier comprises binding sites for the pair of target probes of one of the target probe sets and a plurality of binding sites for an amplifier
Implementation Method 3
each subset of amplifiers comprises a plurality of amplifiers, wherein the amplifiers of a subset of amplifiers comprise a binding site for one of the pre-amplifiers specific for a target probe set and a plurality of binding sites for a label probe
Implementation Method 4
each subset of label probes comprises a plurality of label probes, wherein the label probes in each of the subsets of label probes comprise a label and a binding site for the amplifiers of one of the subsets of amplifiers
Data Source
AI summary
The invention relates to methods of multiplex detection of a plurality of target nucleic acids by contacting a sample comprising a cell with target probe sets that specifically hybridize to target nucleic acids, with pre-amplifiers or pre-pre-amplifiers specific for each target probe set, with amplifiers specific for the pre-amplifiers, and with label probes specific for the amplifiers, resulting in specific labeling of multiple target nucleic acids. The invention also relates to samples, slides and kits relating to detection of multiple target nucleic acids.