RNA Isolation Method Using Denaturing Solution and Protease Digestion
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Solution Overview
Problem
Current RNA isolation methods from biological samples are plagued by biases and inconsistencies due to unstable and uncontrolled purification approaches, leading to variability in yield and RNA integrity, particularly in nuclease-rich and RNA-poor human clinical samples.
Innovation Solution
A method involving a denaturing solution with a reducing agent and detergent, followed by protease treatment, organic extraction, and silica-based solid phase binding, with specific wash solutions to maximize RNA yield and integrity, including the use of chaotropic agents and alcohols to separate and purify RNA effectively.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNA isolation methods are used, then RNA can be extracted from biological samples, but the yield and RNA integrity are inconsistent due to biases introduced by unstable purification approaches
Solution Approach 1:
The patent modifies the chemical parameters of the purification solution by incorporating specific reducing agents (beta-mercaptoethanol, dithiothreitol) and chaotropic agents (guanidinium thiocyanate, guanidinium chloride) at optimized concentrations. These parameter changes create a controlled denaturing environment that stabilizes RNA and prevents degradation, leading to consistent yield and integrity across different samples and extraction batches.
Solution Approach 2:
The patent introduces an intermediary purification solution that acts as a mediator between the biological sample and the final RNA product. This solution contains a specific combination of reducing agents, chaotropic agents, and detergents that facilitate controlled denaturation of proteins while protecting RNA from degradation. The intermediary solution standardizes the extraction process, reducing variability introduced by direct conventional methods.
2Productivity
If standard purification procedures are applied, then RNA can be isolated, but interphase formation occurs during centrifugation requiring higher forces and longer times
Solution Approach 1:
The purification solution acts as an intermediary that modifies the physical-chemical properties of the extraction mixture. By incorporating specific concentrations of chaotropic agents and reducing agents, the solution prevents emulsion formation and interphase trapping during centrifugation. This allows for faster phase separation at lower centrifugal forces, improving productivity while simplifying the centrifugation requirements.
Solution Approach 2:
The patent changes the density and viscosity parameters of the purification solution by optimizing the concentrations of chaotropic agents and detergents. These parameter changes enhance the phase separation characteristics of the extraction mixture, allowing for rapid clarification at lower centrifugal forces and reducing the time required for interphase formation, thus improving overall processing speed.
3Manufacturing precision
If conventional extraction methods are used, then RNA can be recovered, but DNA contamination remains a problem
Solution Approach 1:
The patent optimizes the pH and ionic strength parameters of the purification solution to selectively favor RNA binding to the silica column while leaving DNA in the aqueous phase. By adjusting these parameters and using specific reducing agents, the method enhances the differential binding properties, allowing for effective separation and purification of RNA free from DNA contamination.
Solution Approach 2:
The purification solution serves as an intermediary that facilitates selective RNA extraction. The combination of reducing agents and chaotropic agents in the solution creates conditions where RNA is preferentially bound to the silica column while DNA remains in solution. This intermediary mechanism enables effective removal of DNA contamination during the washing steps, improving RNA 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 method significantly improves RNA intactness and recovery by reducing interphase formation, allowing for lower centrifugal forces and faster phase separation, maximizing RNA recovery while minimizing DNA contamination, thus providing a more reliable and consistent RNA isolation process.
Implementation Method 1
contacting the biological fluid with a denaturing solution comprising at least one of reducing agent and detergent
Implementation Method 2
denaturing solution comprising at least one of reducing agent and detergent
Implementation Method 3
contacting the biological fluid mixture with a protease to form a protease treated biological fluid mixture
Implementation Method 4
contacting the protease treated biological fluid mixture with an organic extraction solution, forming a solution having an aqueous phase containing the RNA and an organic phase
Implementation Method 5
binding the RNA to a silica based solid phase by contacting the aqueous phase with said silica based solid phase
Implementation Method 6
contacting the silica based solid phase with a first wash solution comprising alcohol, chaotropic agent, and reducing agent
Implementation Method 7
eluting the RNA from the silica based solid phase comprising contacting the silica based solid phase with an aqueous solution to provide isolated RNA
Data Source
AI summary
The disclosure provides methods for isolating nucleic acids from a biological fluid. In one aspect, the disclosure provides a method for isolating RNA. In another aspect, the disclosure provides a method for isolating DNA. In one aspect, the methods described herein utilize a protocol that combines a detergent-based initial denaturation, protease digestion, and organic extraction followed by column purification that maximizes RNA/DNA yield and preserves RNA/DNA integrity. In yet another aspect, the disclosure provides a kit for isolating RNA and/or DNA.