Non-formalin Fixation for Biomolecule Integrity
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Solution Overview
Problem
Current methods for stabilizing biological samples, especially non-formalin-fixed samples, fail to effectively preserve the integrity of biomolecules such as RNA and DNA for molecular analysis, leading to degradation and incomplete extraction due to the use of crosslinking agents like formaldehyde, which are also toxic and unsuitable for molecular biology processes.
Innovation Solution
A method involving the use of non-formalin fixation techniques, such as those using Carnoy's, Methacarn, Umfix, Finefix, or PAXgene Tissue Kit, followed by quick staining and de-waxing, allows for the visualization and isolation of biomolecules by mounting the sample on a support, treating with a sample treatment agent, staining for a short duration, and then isolating RNA from the target cells or tissue.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If formalin-based crosslinking fixation is used to stabilize biological samples, then morphological preservation is improved, but biomolecule integrity and extractability deteriorate
Solution Approach 1:
The patent removes formalin and other crosslinking agents from the fixation process, extracting the harmful element that causes biomolecule degradation. Instead, it uses alternative fixatives that do not crosslink proteins, thereby preserving biomolecule integrity while still achieving morphological stabilization.
Solution Approach 2:
The patent changes the chemical parameters of the fixation process by switching from crosslinking agents (formalin) to non-crosslinking alternatives (such as alcohol-based fixatives or protease-based systems). This parameter change eliminates the harmful crosslinking reaction while maintaining the stabilizing function needed for morphological preservation.
2Stability of the object's composition
If formalin fixation is applied to stabilize biomolecules, then sample stabilization is improved, but extractability of nucleic acids and proteins deteriorates
Solution Approach 1:
The patent extracts and removes formalin from the fixation protocol, eliminating the substance that prevents biomolecule extraction. By using alternative non-crosslinking fixatives, the method achieves stabilization without creating a barrier to subsequent extraction processes.
Solution Approach 2:
The patent introduces intermediary substances (alternative fixatives that do not crosslink) that mediate between the need for stabilization and the need for extractability. These intermediaries provide the stabilizing function without the harmful crosslinking effect that blocks extraction.
3Stability of the object's composition
If crosslinking stabilizers like formalin are used, then tissue preservation is improved, but toxicity and suitability for molecular biology deteriorate
Solution Approach 1:
The patent converts the harmful crosslinking mechanism into a beneficial non-crosslinking alternative. By replacing formalin with fixatives that work through different mechanisms (such as protein precipitation or enzymatic stabilization), it maintains tissue preservation while eliminating toxicity and molecular biology incompatibility.
Solution Approach 2:
The patent changes the chemical mechanism parameters of tissue preservation from crosslinking to non-crosslinking methods. This parameter change maintains the stabilizing effect on tissue morphology while removing the harmful toxicity and incompatibility with molecular biology techniques.
4Reliability
If rapid staining is performed to minimize RNA degradation, then RNA integrity is improved, but staining quality and target cell identification deteriorate
Solution Approach 1:
The patent optimizes the staining parameters by using highly sensitive stains that require minimal incubation time. By changing the stain concentration, type, or application method, it achieves sufficient target cell identification within seconds to minutes, thereby preserving RNA integrity while maintaining detection capability.
Solution Approach 2:
The patent replaces prolonged mechanical staining processes with rapid alternative staining methods. This substitution maintains the ability to identify target cells while significantly reducing the time the RNA is exposed to potential degradation, thereby preserving RNA integrity.
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 approach enables the preservation of biomolecules in a life-like state, allowing for effective extraction and analysis of RNA and DNA from non-formalin-fixed samples, maintaining their integrity and enabling downstream applications like quantitative RT-PCR, while avoiding the toxicity and limitations of formalin-based methods.
Implementation Method 1
the biomolecule status of a biological material taken out from its natural environment alters increasingly, the longer the elapsed time between the sample collection and its analysis. In particular the ribonucleic acids (RNA) degrade quickly due to ubiquitous RNases.
Implementation Method 2
staining at least one part of the fixed biological sample mounted on at least one support for not more than 60 seconds with at least one cell or tissue staining agent
Implementation Method 3
isolating the biomolecules from the separated cells or tissue
Data Source
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AI summary
The present invention relates to a method for determination of target cells or tissue for isolating or extracting biomolecules from fixed biological samples, the preparation of a sample in a method for extracting, isolating and/or purifying biomolecules from a fixed biological sample as well as to a kit for visualizing target cells or tissue in a fixed biological sample for extracting, isolating and/or purifying biomolecules from said target cells or tissue.