FFPET Staining with High Salt for RNA Integrity
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Solution Overview
Problem
The challenge in combining laser capture micro-dissection (LCM) with RT-qPCR analysis of formalin-fixed, paraffin-embedded tissue sections is the limited amount of material and significant RNA degradation during the immuno-histochemical staining process, leading to unreliable gene expression analysis results.
Innovation Solution
Incorporating high salt concentrations of 0.5 to 3.0 M sodium chloride in the staining method for formalin-fixed, paraffin-embedded tissue sections to prevent RNA degradation, allowing for improved RNA isolation and quality suitable for gene expression analyses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If classical immuno-histochemical staining procedure is used with extended incubation times, then staining quality is improved, but RNA degradation increases significantly
Solution Approach 1:
The patent applies parameter changes by modifying the chemical composition of the staining buffer, specifically adding sodium chloride at concentrations of 0.5 to 3.0 M to the immuno-histochemical staining solution. This parameter change protects RNA from degradation during extended antibody incubation periods while maintaining staining quality, thereby resolving the contradiction between achieving good staining results and preserving RNA integrity.
2Reliability
If ultra-fast staining procedure is used to minimize RNA degradation, then RNA integrity is improved, but staining quality deteriorates due to insufficient antibody incubation time
Solution Approach 1:
The patent modifies the staining buffer composition by incorporating high concentrations of sodium chloride (0.5 to 3.0 M), which creates a protective environment for RNA during extended incubation periods. This parameter change enables the use of conventional antibody incubation times (e.g., overnight at 4°C or several hours at room temperature) without suffering from RNA degradation, thus achieving both good staining quality and RNA integrity.
3Adaptability or versatility
If FFPET tissue is used instead of fresh frozen tissue, then availability and archive utility are improved, but RNA degradation increases due to fixation and storage processes
Solution Approach 1:
The patent applies parameter changes by introducing high concentrations of sodium chloride (0.5 to 3.0 M) into the immuno-histochemical staining buffer used on FFPET sections. This modification protects the already fixed and stored RNA in the formalin-fixed, paraffin-embedded tissue from further degradation during the staining process, enabling reliable RNA isolation and gene expression analysis from archived FFPET samples that would otherwise be unsuitable due to RNA degradation.
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
The use of high salt concentrations in the staining process significantly reduces RNA degradation, enabling the isolation of high-quality RNA from FFPET sections, which is essential for reliable RT-qPCR analyses and gene expression studies.
Implementation Method 1
heating the tissue section mounted on the solid support to retrieve epitopes at 50 to 70° C. for 12 to 24 h, and c) staining the tissue section mounted on the solid support, wherein at least step e) is performed in the presence of 0.5 to 3.0 M sodium chloride
Implementation Method 2
heating the tissue section mounted on the solid support to retrieve epitopes at 50 to 70° C. for 12 to 24 h
Data Source
AI summary
The present invention refers to a method for immuno-histochemical staining of a formalin-fixed, paraffin-embedded tissue section comprising the steps of a) providing a solid support, b) mounting the formalin-fixed, paraffin-embedded tissue section onto the solid support, c) removing the paraffin from the formalin-fixed, paraffin-embedded tissue section, d) heating the tissue section mounted on the solid support to retrieve epitopes at 50 to 70° C. for 12 to 24 h, and e) staining the tissue section mounted on the solid support, wherein at least step e) is performed in the presence of 0.5 to 3.0 M sodium chloride. The present invention further refers to a kit for performing the method.