RNA Isolation Using Cationic Detergent to Reduce DNA Contamination
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Solution Overview
Problem
Current RNA isolation methods using phenol-chloroform extraction leave residual DNA contaminations in purified RNA, which can distort RNA-dependent RT-PCR results and require additional costly purification steps like DNase treatment or nucleic acid binding phases.
Innovation Solution
Incorporating a cationic detergent into the sample before phase separation during phenol-chloroform extraction, allowing DNA to be efficiently removed from the aqueous phase and concentrated in the interphase or organic phase, thereby reducing DNA contaminations in the isolated RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If phenol-chloroform extraction is used for RNA isolation, then RNA purity is improved, but residual DNA contaminations remain in the purified RNA
Solution Approach 1:
The invention changes the chemical composition parameter of the extraction system by adding cationic detergent to the phenol-chloroform extraction mixture. This parameter change modifies the phase separation behavior, causing DNA to preferentially partition into the interphase or organic phase rather than remaining in the aqueous phase with RNA, thereby reducing DNA contamination in the purified RNA
Solution Approach 2:
The cationic detergent acts as an intermediary substance that facilitates the separation of DNA from RNA during phase separation. The detergent interacts with both nucleic acids and the phenol-chloroform system, modifying their distribution between phases and enabling selective removal of DNA without compromising RNA purity
2Object-affected harmful factors
If additional purification steps like DNase treatment or nucleic acid binding phases are used, then DNA contaminations are reduced, but costs and handling steps increase
Solution Approach 1:
The invention merges the DNA removal function with the existing phenol-chloroform extraction process by adding cationic detergent. This combines multiple functions (RNA extraction, phase separation, and DNA removal) into a single integrated procedure, eliminating the need for separate DNase treatment or additional nucleic acid binding steps while maintaining effective DNA contamination reduction
Solution Approach 2:
The invention extracts the DNA removal function from the separate purification steps and integrates it into the primary phenol-chloroform extraction process. By taking out the need for additional purification steps and incorporating DNA removal into the main extraction workflow, the method simplifies the overall procedure while achieving the same or better DNA contamination reduction
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 effectively reduces DNA contaminations in RNA, eliminating the need for additional purification steps and maintaining RNA quality, while being cost-efficient and simple to implement.
Implementation Method 1
The homogenate is subjected to phase separation by adding a water-insoluble organic solvent such as chloroform. Following centrifugation, the mixture separates into an aqueous phase containing RNA and an interphase and organic phase which contain DNA and proteins.
Implementation Method 2
the addition of at least one cationic detergent to a sample that is treated by the addition of an acidic denaturing composition comprising a chaotropic agent and phenol considerably decreases the amount of DNA in the isolated RNA
Data Source
AI summary
The present invention pertains to a method of isolating RNA from a sample comprising RNA, and DNA, comprising: a) adding an acidic denaturing composition comprising a chaotropic agent and phenol to the sample; b) adding a water-insoluble organic solvent and separating the resulting phases thereby forming a multi-phase mixture comprising an aqueous phase, optionally an interphase and an organic phase, wherein the RNA is concentrated in said aqueous phase and DNA and proteins are concentrated in said organic phase and/or in said interphase; and c) isolating said RNA from said aqueous phase, wherein at least one cationic detergent is added before separating the phases. It was found that the addition of at least one cationic detergent considerably reduces the amount of DNA in the aqueous, RNA containing phase. Therefore, the present invention allows to easily isolate pure RNA which comprises considerably less DNA contaminations.