FFPE Nucleic Acid Extraction Using Hexadecane Deparaffinization
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Solution Overview
Problem
Current methods for extracting nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissue samples are challenging due to the use of toxic solvents, prolonged protease digestion, and chemical modification of nucleic acids, resulting in low yields and degraded products.
Innovation Solution
The method involves using a wax-solubilizing organic solvent, such as hexadecane, to dissolve the wax from FFPE samples, followed by the addition of alcohol to form a precipitate containing nucleic acids. This is then washed and combined with a lysis buffer containing non-ionic detergents and RNase inhibitors to isolate the nucleic acids using beads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If xylene is used to deparaffinize FFPE samples, then the wax is effectively removed, but toxic chemical exposure and environmental harm occur
Solution Approach 1:
The patent replaces toxic xylene with less harmful alternative solvents such as toluene, xylene substitutes, or specialized deparaffinization buffers that maintain effective wax removal while eliminating or reducing toxic chemical exposure and environmental harm
Solution Approach 2:
The patent introduces intermediary substances such as specialized buffers or alternative organic solvents that serve as mediators between the FFPE sample and the deparaffinization process, achieving effective wax removal without direct contact with highly toxic chemicals
2Quantity of substance
If prolonged protease digestion is used to extract nucleic acids, then nucleic acid release is improved, but processing time increases and nucleic acid degradation occurs
Solution Approach 1:
The patent modifies parameters such as protease concentration, temperature, and pH conditions to optimize the digestion process, achieving high nucleic acid yield in reduced time while preventing degradation through controlled reaction conditions
Solution Approach 2:
The patent performs preliminary steps such as optimized deparaffinization and tissue disruption before protease digestion, preparing the sample in advance to enable faster and more efficient nucleic acid release during the digestion step
3Quantity of substance
If prolonged protease digestion is used, then nucleic acid release is improved, but nucleic acid degradation increases
Solution Approach 1:
The patent carefully controls parameters including temperature, pH, and protease concentration during digestion to maximize nucleic acid release while maintaining integrity, and uses stabilizing agents in the extraction buffer to prevent degradation
Solution Approach 2:
The patent optimizes the digestion process to complete nucleic acid release in the minimum necessary time, rushing through the critical period where degradation could occur while still achieving sufficient yield
4Quantity of substance
If high salt solutions are used for nucleic acid isolation, then nucleic acid precipitation is improved, but chemical modification and fragmentation increase
Solution Approach 1:
The patent uses alternative precipitation methods such as alcohol precipitation with optimized conditions, or silica-based purification, replacing harsh high salt solutions to achieve effective nucleic acid recovery without chemical modification or fragmentation
Solution Approach 2:
The patent introduces intermediary substances such as alcohol or silica particles that mediate the precipitation or binding process, enabling nucleic acid isolation without direct exposure to damaging high salt conditions
5Manufacturing precision
If multiple extraction steps are used, then nucleic acid purity is improved, but process complexity increases
Solution Approach 1:
The patent combines multiple extraction steps into integrated procedures, such as combining deparaffinization with initial lysis, or merging purification steps into a single column-based or magnetic bead-based process, maintaining high purity while reducing overall complexity
Solution Approach 2:
The patent employs universal reagents or columns that can perform multiple functions (deparaffinization, lysis, and purification) in a single step, reducing the number of separate operations needed while maintaining nucleic acid purity
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 results in higher yields of intact nucleic acids, reduced degradation, and an increased number of unique RNA species, enhancing the quality and quantity of nucleic acids suitable for sequencing and expression analysis.
Implementation Method 1
The sample is combined with a wax-solubilizing organic solvent to form a mixture, wherein substantially all (>95%, preferably >98%) of the wax is in the liquid phase
Implementation Method 2
An alcohol is then added to the mixture. A precipitate or solid phase is formed before or after the adding of the alcohol that contains nucleic acids from the sample
Implementation Method 3
The organic solvent and the alcohol are then separated from the precipitate, such that at least 90%, preferably 97%, or all of the solvent or alcohol originally added to the sample is removed. The means for removal may be centrifugal separation, gravity precipitation, filtration, or column separation
Data Source
Figure 1A~1B
Figure 2
Figure 3A~3B
AI summary
Disclosed herein is a method and kit thereof for isolating nucleic acids from wax-embedded samples using hexadecane as the solvent to dissolve the sample, precipitating and washing the extracted nucleic acids using ethanol, and dissolving the nucleic acids in a lysis buffer that includes NP40 and SDS. By implementing the reagents and techniques described in this disclosure, the user can obtain a product that has better yield, less degradation, and contains more unique mRNA transcripts for subsequent sequencing and analysis.