RNA Recovery from Fixed Cells via Protein-Nucleic Acid Dissociation
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Solution Overview
Problem
Current methods for analyzing intracellular protein and RNA in fixed cells are incompatible due to cross-linking issues, making simultaneous analysis of proteins and nucleic acids challenging for single cell genomics.
Innovation Solution
A method involving fixing and permeabilizing cells, followed by using oligonucleotide barcodes and specific reagents to dissociate protein-nucleic acid complexes, allowing for the simultaneous analysis of proteins and nucleic acids by hybridization and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If protein-compatible fixation methods are used to detect and analyze intracellular protein targets, then protein analysis is improved, but RNA recovery is reduced due to cross-linking
Solution Approach 1:
The patent introduces an intermediary agent (such as formic acid, guanidine, or other denaturing agents) that mediates between the fixed cells and the RNA extraction process. This intermediary disrupts the protein-RNA cross-links formed during fixation without completely destroying the cellular structure, thereby enabling RNA recovery while maintaining protein analysis capability
Solution Approach 2:
The patent employs parameter changes by adjusting pH, temperature, and chemical concentration during the RNA extraction process from fixed cells. By optimizing these parameters (e.g., using acidic conditions with formic acid or high concentrations of denaturing agents), the method reverses fixation-induced cross-linking effects and restores RNA accessibility for analysis
2Stability of the object's composition
If cross-linking fixatives are used to preserve cellular structure, then cell morphology is maintained, but simultaneous RNA and protein analysis becomes incompatible
Solution Approach 1:
The patent applies preliminary action by treating fixed cells with RNA-preserving reagents (such as formic acid or guanidine) before proceeding with RNA extraction. This preliminary treatment prevents permanent cross-linking damage and maintains RNA integrity, enabling subsequent multi-omics analysis while preserving cell structure
Solution Approach 2:
The patent converts the harmful effect of cross-linking fixatives into a benefit by using the same fixed cellular structure as a starting point and then applying reversal agents that specifically target the cross-links. The fixation-induced cross-links are converted from obstacles into manageable features that can be selectively reversed, enabling both structural preservation and molecular analysis
3Loss of substance
If standard RNA extraction methods are used on fixed cells, then RNA can be recovered, but protein analysis capability is lost due to cross-linking
Solution Approach 1:
The patent applies segmentation by separating the RNA extraction and protein analysis workflows into distinct, optimized pathways. The RNA extraction pathway uses specific reagents and conditions tailored for recovering RNA from fixed cells, while the protein analysis pathway uses separate reagents and conditions, allowing both analyses to be performed on the same fixed cell sample without interference
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 effective preservation and recovery of RNA and protein analysis in fixed cells, facilitating accurate single cell omics analysis by dissociating protein-nucleic acid complexes and enabling simultaneous protein and RNA profiling.
Implementation Method 1
contacting a plurality of oligonucleotide barcodes with the copies of the nucleic acid target and the protein target-binding reagent specific oligonucleotides for hybridization
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for preserving and/or recovering RNA from fixed and/or permeabilized cells, for example for single cell analysis. In some embodiments, the fixed and/or permeabilized cells are for measuring intracellular target (e.g., protein) expression.