Cold Formalin Fixation for Tissue RNA Preservation
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Solution Overview
Problem
Current methods for fixing organic tissue samples using formalin result in poor preservation of RNA, making it unsuitable for gene expression analysis, particularly as only short sequences can be obtained from formalin-fixed paraffin-embedded (FFPE) tissues, which are fragmented and degraded.
Innovation Solution
A two-step cold formalin fixation method involving immersion of tissue samples in a formalin solution at 2°C to 5°C for 18-30 hours followed by immersion in cold 95% ethanol at the same temperature for 2-4 hours, which effectively preserves both histological and RNA integrity, allowing for longer mRNA sequences to be obtained for gene expression analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If tissue samples are fixed in formalin at room temperature for several hours to achieve optimal histological preservation, then morphological and antigenic properties are well preserved, but RNA is heavily degraded and fragmented
Solution Approach 1:
The patent applies parameter changes by lowering the fixation temperature from room temperature to 2-10°C and extending the fixation time to 18-30 hours. This temperature-time parameter modification slows down the degradation processes while maintaining effective protein crosslinking, thereby preserving both histological structure and RNA integrity simultaneously.
Solution Approach 2:
The patent segments the fixation process into two distinct steps: (1) cold formalin fixation for 18-30 hours to preserve morphology and RNA, followed by (2) cold alcohol dehydration for 2-4 hours to remove water and prepare for paraffin embedding. This segmentation allows each step to be optimized for its specific function without compromising the other.
2Reliability
If tissue samples are fixed in formalin for extended periods to improve RNA preservation, then RNA degradation is reduced, but the process becomes time-consuming and complex
Solution Approach 1:
The patent divides the extended fixation process into two sequential steps with specific time allocations: Step 1 (cold formalin fixation) occupies 18-30 hours for RNA and morphology preservation, while Step 2 (cold alcohol dehydration) occupies 2-4 hours for rapid water removal. This segmentation optimizes time utilization by performing different functions in separate phases.
Solution Approach 2:
By changing the temperature parameter to cold conditions (2-10°C) during the extended fixation period, the patent slows down degradation reactions, allowing better RNA preservation within the given time frame. The cold temperature acts as a protective parameter that reduces the rate of RNA degradation while maintaining fixation effectiveness.
3Shape
If standard formalin fixation is used to maintain tissue structure, then cellular morphology is preserved, but gene expression analysis becomes difficult due to RNA fragmentation
Solution Approach 1:
The patent modifies the fixation parameters by using cold formalin (2-10°C) instead of room temperature formalin, and extends the fixation time to 18-30 hours. These parameter changes create a protective environment that preserves both cellular morphology and RNA integrity, enabling subsequent gene expression analysis without the usual RNA fragmentation problems.
Solution Approach 2:
The patent segments the fixation process into morphology-preserving formalin fixation followed by alcohol dehydration, allowing the tissue to undergo complete formalin fixation for structure preservation before the dehydration step. This segmentation ensures that morphology is optimized in the first step while RNA is protected during the extended fixation period.
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 achieves optimal preservation of mRNA sequences longer than 350 nucleotides, enabling effective gene expression profiling from FFPE tissues, which was previously not possible with shorter sequences from FFPE tissues.
Implementation Method 1
Formalin is a protein-crosslinking fixing agent
Implementation Method 2
immersing the tissue in a solution of Formalin at a temperature of between 2°C and 10°C, preferably 2°C to 5°C
Implementation Method 3
immersing the tissue in a cold fixation dehydrating agent, preferably an alcohol such as e.g. ethanol, especially 95% ethanol, for a second time period
Data Source
Figure 1a~1d
Figure 2
Figure 3
AI summary
The invention proposes a method and a system for a two-step fixation of organic tissue samples (2), the method comprising at least the steps of: a.) immersing the tissue (2) in a solution of Formalin (4) at a temperature of between 2°C and 10°C, preferably 2°C to 5°C, for a first time period (A), and b.) immersing the tissue (2) in a cold fixation dehydrating agent (6), preferably 95% Ethanol, for a second time period (B).