FFPE Nucleic Acid Extraction Without Organic Solvents
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Solution Overview
Problem
Current methods for nucleic acid extraction from formalin-fixed paraffin-embedded (FFPE) tissues are cumbersome, time-consuming, and prone to sample degradation due to the use of hazardous solvents like xylene, leading to non-quantitative harvests and inaccurate RNA quantification due to fragmentation and base modifications caused by formalin fixation.
Innovation Solution
A method involving the suspension of FFPE samples in an aqueous solution at elevated temperatures to release nucleic acids without denaturing conditions, followed by physical separation from hydrophobic components, allowing for accurate mRNA copy number determination using ribosomal DNA normalization and degraded in vitro RNA standards to account for fragmentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional organic extraction methods using xylene and graded alcohols are used for dewaxing, then paraffin removal is achieved, but the process becomes time-consuming, laborious, and causes sample degradation
Solution Approach 1:
The patent changes the fundamental parameter of the dewaxing approach by eliminating organic solvents entirely and using a water-based system with detergents and enzymes. This transforms the extraction chemistry from organic to aqueous, enabling paraffin removal without the time-consuming repeated washing steps required by traditional methods while preventing nucleic acid degradation from harsh chemical exposure
Solution Approach 2:
The patent replaces the mechanical phase extraction process (repeated centrifugation, aspiration, and tube transfers) with a simplified aqueous extraction system. The water-based detergent solution dissolves paraffin directly, and nucleic acids are recovered through simple precipitation or filtration, eliminating the complex mechanical operations that consume time and risk sample loss
2Reliability
If repeated vortexing and harsh solvent exposure are used for dewaxing, then paraffin is removed, but sample degradation increases
Solution Approach 1:
The patent converts the harmful effect of paraffin (which resists aqueous solutions) into a benefit by using water-soluble detergents that specifically target and dissolve hydrophobic paraffin. The detergent molecules emulsify paraffin droplets, allowing gentle removal without harsh solvents or vigorous mechanical treatment, thus protecting nucleic acid integrity while achieving complete dewaxing
Solution Approach 2:
The patent introduces detergent as an intermediary substance that mediates between the hydrophobic paraffin and the aqueous extraction environment. The detergent's amphiphilic structure allows it to solubilize paraffin in water, enabling gentle extraction that avoids the degradation caused by direct exposure to harsh organic solvents and mechanical stress
3Measurement precision
If standard analytical techniques are used to evaluate extracted nucleic acids, then quantification is performed, but errors result due to fragmentation and degradation
Solution Approach 1:
The patent performs preliminary normalization using ribosomal DNA (rDNA) copies as an internal reference before final quantification. By measuring rDNA content in the same sample and using it to normalize the target nucleic acid measurements, the method compensates for variations in extraction efficiency and fragmentation, providing accurate quantification even when samples are degraded
Solution Approach 2:
The patent implements a feedback mechanism by using degraded in vitro transcribed RNA standards that mimic the fragmentation pattern of FFPE samples. These standards are processed through the same extraction and analysis pipeline, allowing the system to self-calibrate and correct for degradation effects, thereby achieving accurate quantification despite sample fragmentation
4Duration of action of stationary object
If formalin fixation is used for tissue preservation, then clinical specimens are preserved for long-term storage, but RNA fragmentation and base modifications occur
Solution Approach 1:
The patent applies partial reversal of formalin fixation effects by using prolonged enzymatic treatment with proteinase K and specific buffer conditions that partially reverse cross-linking. This partial action is sufficient to release fragmented RNA from protein-DNA complexes without requiring complete reversal of fixation, thus maintaining the benefit of long-term preservation while enabling adequate RNA extraction and quantification
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 simplifies and enhances the accuracy of nucleic acid extraction and quantification from FFPE samples, reducing handling errors and improving the reliability of mRNA copy number analysis by avoiding chemical extractions and accounting for sample degradation.
Implementation Method 1
suspending the sample in an aqueous solution, incubating the suspension at a temperature higher than 40° C. so that the hydrophobic component melts and the nucleic acid is released from the cells into the aqueous solution
Implementation Method 2
The suspension can be centrifuged in a microcentrifuge at 16,000 × g for 2 min, and the supernatants removed
Data Source
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AI summary
This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.