RNA Extraction from Urine Microvesicles Using Enzyme Inhibition
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Solution Overview
Problem
Current methods for extracting high-quality nucleic acids from microvesicles in biological samples, such as urine, face challenges in achieving consistent and reliable results due to adverse factors like excessive DNA and RNase contamination, which affect the integrity and yield of extracted RNA.
Innovation Solution
A method involving pre-processing of urine samples through filtration and ultracentrifugation or filtration concentration, followed by washing and treatment with RNase inhibitors and proteases to enhance nucleic acid extraction, ensuring the detection of 18S and 28S rRNA with a preferred ratio and high RNA Integrity Number (RIN), and using extraction enhancement agents like RNase inhibitors and proteases to mitigate adverse factors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If ultracentrifugation or filtration concentration is used to obtain microvesicle fraction, then microvesicles can be isolated from urine sample, but excessive DNA and RNase contamination occurs which affects RNA integrity and yield
Solution Approach 1:
The patent applies preliminary action by treating the microvesicle fraction with DNase and RNase inhibitors before RNA extraction. This pre-treatment step removes harmful contaminants (excessive DNA and RNase) before they can degrade the RNA, thereby improving RNA integrity and yield while maintaining effective microvesicle isolation
Solution Approach 2:
The patent extracts and removes harmful factors by using DNase to digest excessive DNA and RNase inhibitors to neutralize ribonuclease contamination. This extraction of harmful substances from the microvesicle fraction prevents RNA degradation and improves the quality of extracted RNA
2Quantity of substance
If RNA extraction is performed from microvesicle fraction, then RNA can be obtained for analysis, but RNA integrity and yield are insufficient for reliable biomarker analysis
Solution Approach 1:
The patent performs preliminary protection of RNA by adding RNase inhibitors and proteases to the microvesicle fraction before extraction. This prevents RNA degradation during the extraction process, ensuring high RNA integrity and yield suitable for reliable biomarker analysis
Solution Approach 2:
The patent uses RNase inhibitors and proteases as intermediary substances that protect RNA from degradation. These intermediaries bind to or inhibit RNases and proteases, creating a protective environment that maintains RNA integrity throughout the extraction process
3Productivity
If filtration through 0.8μm filter is used for pre-processing, then urine sample can be pre-processed efficiently, but some microvesicles may be lost reducing extraction efficiency
Solution Approach 1:
The patent optimizes the filtration parameters by using a 0.8μm filter, which balances between removing large debris (improving processing efficiency) and retaining microvesicles (maintaining recovery). This parameter optimization ensures both efficient pre-processing and adequate microvesicle recovery for subsequent analysis
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 the yield and integrity of RNA from microvesicles, achieving RNA Integrity Numbers of 5 or higher for low-protein samples and 3 or higher for high-protein samples, with increased detection of 18S and 28S rRNA, making the extracted nucleic acids suitable for various applications, including biomarker analysis.
Implementation Method 1
pre-processing the urine sample by filtration through a 0.8μm filter
Implementation Method 2
obtaining a microvesicle fraction from the pre-processed urine sample by ultracentrifugation
Implementation Method 3
treatment with RNase inhibitors and proteases to enhance nucleic acid extraction
Data Source
Figure 1a~1f
Figure 2
Figure 3~4
AI summary
A method for extracting high quality nucleic acids from a biological sample are disclosed The extractions obtained by the methods described herein are characterized by high yield and high integrity, making the extracted nucleic acids useful for various applications in which high quality nucleic acid extractions are preferred, e g, a diagnosis, prognosis or therapy evaluation for a medical condition