Cell Permeabilization for Preserving Intracellular RNA Integrity

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Solution Overview

Problem

Existing methods for stabilizing intracellular RNA in permeabilized cells, such as formaldehyde fixation, cause chemical modifications that compromise nucleic acid integrity, limiting the efficiency and accuracy of downstream gene expression analysis.

Innovation Solution

A method involving alcohol fixation followed by salt solution treatment to inactivate enzymes and maintain membrane integrity, then using a buffer to preserve RNA integrity, allowing permeabilization of cells for single-cell RNA sequencing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If formaldehyde fixation is used to preserve intracellular RNA, then cell morphology is preserved, but RNA integrity is compromised due to chemical modifications

Engineering Contradiction:

Improvecell morphology preservationVSAvoidRNA integrity

Core Design Contradiction:

Stability of the object's compositionVSReliability

Solution Approach 1:

The invention changes the chemical parameters of fixation by using alcohol-based fixatives (methanol or ethanol) instead of formaldehyde, and optimizes fixation time and temperature parameters to preserve RNA integrity while maintaining cell morphology

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention extracts and removes cross-linking agents and their harmful chemical modifications from the fixation process, using alternative fixation mechanisms that do not form cross-links between nucleic acids and proteins

Inventive Principle:

Principle #2Taking out (Extraction)

2Ease of operation

If organic solvents are used for simultaneous fixation and permeabilization, then cell permeability is improved, but RNA integrity is damaged due to protein coagulation

Engineering Contradiction:

Improvecell permeabilityVSAvoidRNA integrity

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The invention segments the fixation and permeabilization processes into separate steps, first fixing cells with alcohol to preserve RNA, then applying controlled permeabilization to allow antibody access while minimizing RNA damage

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces controlled permeabilization agents as intermediaries that facilitate antibody access to intracellular targets without causing the extensive protein coagulation and RNA damage associated with strong organic solvent treatment

Inventive Principle:

Principle #24Intermediary (Mediator)

3Stability of the object's composition

If cross-linking agents are used to fix cells, then cell structure is preserved, but downstream RNA isolation and detection efficiency is reduced

Engineering Contradiction:

Improvecell structure preservationVSAvoidRNA isolation efficiency

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The invention changes the fundamental mechanism of fixation from cross-linking to precipitation and denaturation using alcohol-based fixatives, which preserve cell structure without forming cross-links that would interfere with downstream RNA isolation and detection

Inventive Principle:

Principle #35Parameter changes

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

Enhances RNA preservation and permeability, enabling strong intracellular protein staining and single-cell analysis with improved RNA quantity and quality, particularly in immune cells.

Implementation Method 1

contacting the cell with a solution of alcohol under conditions that fixes the cell

Methodology Applied

Scientific EffectProtein denaturation:

Implementation Method 2

the salt of the solution is at a concentration which inactivates enzymes of the cell whilst maintaining the integrity of the cell membrane of the cell

Methodology Applied

Scientific EffectEnzyme inactivation: