In Situ Gene Expression Benchmarking With Defined Cell Pellets
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Solution Overview
Problem
Existing in situ analyte detection and sequencing platforms face variability in performance assessment and comparison due to dependence on biological samples and specific detection platforms, making it challenging to evaluate their effectiveness reliably.
Innovation Solution
A performance test using a standardized test biological sample with defined cell populations, such as a mixture of Jurkat and Raji cells, involves hybridizing probes specific to each population's gene expression, allowing for detection and comparison of RNA transcripts, and analyzing sensitivity and specificity through expected expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If in situ assays are performed in living cells to assess drug effects on gene expression, then biological relevance and applicability to complex diseases are improved, but technical challenges including cell permeability, assay sensitivity, and data interpretation complexity worsen
Solution Approach 1:
The patent uses cell-permeable oligonucleotide probes as intermediaries that can penetrate living cell membranes and hybridize to target transcripts within the cell. These probes serve as mediators between the extracellular assay environment and the intracellular target molecules, enabling detection without cell lysis while maintaining biological relevance.
Solution Approach 2:
The patent replaces complex mechanical cell lysis and RNA extraction procedures with a simplified chemical hybridization-based detection system. By using cell-permeable probes that directly hybridize to target transcripts in living cells, the mechanical complexity of traditional RNA isolation is substituted with a more straightforward in situ hybridization approach.
2Measurement precision
If traditional RNA isolation and qPCR methods are used to measure gene expression, then measurement precision is improved, but loss of time and complexity of procedures worsen
Solution Approach 1:
The patent performs the hybridization detection step before cell lysis and RNA extraction. By pre-incubating cell-permeable probes with living cells to allow direct hybridization to target transcripts in situ, the assay eliminates the time-consuming sequential steps of RNA isolation, cDNA synthesis, and qPCR amplification required by traditional methods.
Solution Approach 2:
The patent merges multiple traditional assay steps (cell permeabilization, RNA extraction, cDNA synthesis, and qPCR detection) into a single integrated in situ hybridization procedure. The cell-permeable probes combine the functions of cell entry, target recognition, and signal generation, reducing the overall procedure time while maintaining measurement precision.
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
This method provides a robust and reproducible assessment of in situ analyte detection platforms, enabling reliable validation, verification, and comparison of performance across different platforms, with high sensitivity and specificity in detecting RNA transcripts.
Implementation Method 1
The probe is hybridized to a target nucleic acid sequence
Implementation Method 2
The label can be any detectable label, including, but not limited to, a fluorescent label
Data Source
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AI summary
The present disclosure relates in some aspects to methods and compositions for assessing performance of in situ analyte detection and/or platforms (e.g., methods, assays, workflows, and/or instruments for in situ analyte detection and/or sequencing). In some aspects, performance can be assessed for an individual platform, or the performance of two or more different platforms can be compared. In some aspects, assessing performance comprises a standardized in situ analyte detection workflow and analysis on a test biological sample comprising defined cell populations.