FFPE Tissue RNA Retrieval via Pressure Heating
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Solution Overview
Problem
Current methods for preparing formalin-fixed, paraffin-embedded (FFPE) tissue samples for in situ hybridization often result in degraded RNA, fragmented probes, high autofluorescence, and compromised morphological features, leading to poor signal quality and reproducibility issues, especially when trying to achieve standardization across different tissue types.
Innovation Solution
A workflow involving deparaffinization followed by uniform heating and pressurization at 120°C in an atmosphere of 30 psi, which preserves RNA and morphological features, allowing for higher multiplex detection of genes and proteins without the need for enzymatic digestion, and enabling rapid processing suitable for various tissue types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional preparation methods are used for FFPE tissue samples, then the process is simple and quick, but RNA degradation occurs and signal quality deteriorates
Solution Approach 1:
The preparation process is divided into distinct sequential steps: deparaffinization with organic solvents, hydration with alcohol gradients, and heat/pressure treatment. This segmentation allows each step to optimize for its specific function, improving overall RNA preservation and signal quality while maintaining procedural clarity
Solution Approach 2:
The method performs preliminary deparaffinization and hydration treatments before the main hybridization assay. By removing paraffin and restoring tissue hydration in advance, the sample is pre-conditioned to prevent RNA degradation during subsequent steps, ensuring better signal quality without adding complexity to the core assay
2Reliability
If enzymatic digestion is used to enhance RNA accessibility, then probe hybridization efficiency improves, but morphological features and antigenic epitopes are compromised
Solution Approach 1:
The method replaces enzymatic digestion (chemical/biological system) with mechanical heat and pressure treatment. The high temperature (up to 100°C) and pressure (up to 15 psi) physically disrupt tissue architecture to enhance RNA accessibility and probe penetration, achieving hybridization efficiency without compromising morphological features or antigenic epitopes that would be damaged by enzymatic treatment
Solution Approach 2:
The method changes physical parameters (temperature and pressure) to achieve RNA accessibility instead of using enzymatic chemical reactions. By controlling temperature up to 100°C and applying pressure up to 15 psi during a defined time period, the treatment enhances probe hybridization efficiency while preserving tissue morphology and antigenicity that would be compromised by enzymatic digestion
3Reliability
If high temperature heating is applied to retrieve RNA, then RNA accessibility improves, but tissue morphology and antigenic features degrade
Solution Approach 1:
The method introduces pressure (up to 15 psi) as an intermediary factor that works synergistically with heat. The pressure helps maintain tissue structural integrity during high-temperature heating by counteracting thermal expansion and preventing excessive denaturation, thereby enabling effective RNA retrieval while preserving morphological features and antigenic epitopes
Solution Approach 2:
The treatment combines thermal energy and mechanical pressure into a composite treatment regime. This composite approach allows the benefits of high-temperature RNA retrieval to be achieved while the pressure component protects tissue morphology, creating a synergistic effect that improves RNA accessibility without degrading tissue structure or antigenic features
4Productivity
If standard deparaffinization protocols are used, then the process is fast, but RNA integrity and signal consistency deteriorate
Solution Approach 1:
The method performs thorough deparaffinization with organic solvents and systematic hydration with alcohol gradients as preliminary steps before hybridization. This preliminary treatment ensures complete removal of paraffin and proper tissue rehydration, which prevents RNA degradation and ensures consistent signal quality across samples without significantly extending the overall processing time
Solution Approach 2:
The method optimizes parameters during deparaffinization and hydration steps, including solvent composition, incubation times, and temperature control. By carefully adjusting these parameters, the protocol achieves both rapid processing and consistent RNA preservation, improving signal consistency while maintaining high productivity across different tissue types
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 enhances RNA retrieval and preservation, improving signal quality, reducing batch variability, and enabling simultaneous multiomics analysis with consistent results across different tissue samples, including those with low RNA integrity, while eliminating the need for expensive heating equipment.
Implementation Method 1
The slides are then placed in a pressure chamber, where they are uniformly heated to about 120° C. in an atmosphere of 30 psi
Data Source
AI summary
This disclosure provides a technology for optimally retrieving and presenting RNA in tissue samples for analysis by in situ hybridization, simultaneously preserving morphological and antigenic features of the tissues. Formalin-fixed, paraffin-embedded (FFPE) sections of a tissue sample are dried on glass slides, and deparaffinized by incubating in successive mixtures of organic solvents. The slides are then placed in a pressure chamber, where they are uniformly heated to about 120° C. in an atmosphere of 30 psi. After cooling, the slides are prepared for in situ hybridization and other types of analysis. The RNA retrieval process of this disclosure preserves tissue morphology, antigenic epitopes, and other features. The tissue is thereby optimized for a multiomics workflow, and for higher multiplex detection of genes and proteins in the tissue.