Short RNA Extraction via Protein Precipitation and Adsorption
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Solution Overview
Problem
Current methods for purifying short RNA are hindered by the use of toxic chemicals like phenol, incomplete removal of proteins, and difficulties in automation, leading to low yields and contamination issues, especially when trying to isolate short RNA from samples containing long RNA, DNA, and proteins.
Innovation Solution
A method involving protein precipitation with divalent metal ions, followed by adsorption of short RNA onto a solid support and subsequent desorption, allowing for high-yield and high-purity extraction of short RNA while separating long nucleic acids and proteins, using chaotropic reagents and organic solvents to manage the binding and separation processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If phenol/chloroform liquid-liquid extraction is used to remove DNA and protein, then purification is achieved, but health risks increase and automation becomes difficult
Solution Approach 1:
The patent extracts and removes the harmful phenol/chloroform extraction step from the purification process. Instead, it uses selective precipitation of proteins and long nucleic acids using chaotropic reagents and alcohol, followed by size-exclusion chromatography to separate short RNA from other macromolecules, thereby eliminating phenol while maintaining purification quality
Solution Approach 2:
The patent introduces size-exclusion chromatography as an intermediary separation mechanism. This chromatographic method acts as a mediator that separates molecules based on size differences, allowing short RNA to be isolated from proteins and long nucleic acids without requiring toxic phenol extraction
2Productivity
If protein is not completely removed before small RNA precipitation, then precipitation efficiency is maintained, but co-precipitation occurs and further purification becomes difficult
Solution Approach 1:
The patent applies preliminary protein removal by precipitating proteins and long nucleic acids using chaotropic reagents and alcohol before the small RNA precipitation step. This preliminary action ensures that proteins are removed in advance, preventing co-precipitation and making subsequent small RNA purification easier while maintaining high precipitation efficiency
3Manufacturing precision
If manual liquid-liquid extraction is used, then separation is achieved, but automation and sample throughput are limited
Solution Approach 1:
The patent replaces the manual mechanical liquid-liquid extraction process with a chemistry-based selective precipitation system using chaotropic reagents and alcohol, followed by size-exclusion chromatography. This substitution eliminates the need for manual phase separation while maintaining separation quality and enabling automation of the workflow
4Measurement precision
If short RNA is isolated from traces in a large background of long RNA, DNA and protein, then specific activity is increased, but extraction complexity and time increase
Solution Approach 1:
The patent changes the chemical parameters of the solution by adding chaotropic reagents and alcohol to precipitate proteins and long nucleic acids. This parameter change selectively removes the background macromolecules while keeping short RNA in solution, enabling rapid isolation without complex procedures or time loss
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 enables the quantitative and pure extraction of short RNA, along with the separation of long nucleic acids and proteins, reducing contamination and enabling automated processing, thus improving the reliability of RNA and protein analysis in biological samples.
Implementation Method 1
removing the proteins and the long nucleic acids from the solution, at least the proteins being precipitated
Implementation Method 2
adsorbing the short RNA on a solid (first) support after precipitation of the proteins
Implementation Method 3
using chaotropic reagents and organic solvents to manage the binding and separation processes
Data Source
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AI summary
Extracting at least a short RNA comprises: providing a biological solution containing short RNA and proteins and/or long nucleic acids, preferably RNA and DNA; separating the proteins and long nucleic acids from the solution, where the proteins are precipitated; adsorbing the short RNA at a solid (first) carrier after precipitating the proteins; and extracting the short RNA by desorption from the carrier. An independent claim is included for a kit for extracting the short RNA comprising at least a solid carrier for adsorption of nucleic acids, protein precipitating reagent, at least one binding substance for selective adsorption of short RNA at a solid carrier, and a manual with procedural steps.