Phenol-Free RNA Isolation via Metal Cation Precipitation
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Solution Overview
Problem
Current RNA isolation methods, especially from protein-rich samples, face challenges with high alcohol concentrations that lead to protein precipitation and contamination, reducing the yield of small and large RNA, and often require phenol-based extraction which is toxic and inefficient.
Innovation Solution
A phenol-free RNA isolation method using a metal cation-induced protein precipitation step with a low concentration of metal cation precipitant and a pH of 4.0 or less, avoiding organic solvents to achieve a protein-depleted supernatant containing both small and large RNA, allowing for efficient isolation without phenol or proteolytic enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high alcohol concentration (≥45-50%) is used during RNA binding to ensure efficient binding of small RNA to solid phase, then binding efficiency of small RNA is improved, but proteins are precipitated onto the solid phase causing contamination and blocking
Solution Approach 1:
The patent applies preliminary action by performing protein precipitation with metal cations (such as zinc or copper salts) before the RNA binding step. This preliminary protein removal prevents proteins from being present during the high alcohol concentration binding step, thereby eliminating the harmful effect of protein precipitation while maintaining efficient small RNA binding.
Solution Approach 2:
The patent extracts and removes proteins from the sample using metal cation-induced precipitation before proceeding with RNA isolation. This separation of proteins from nucleic acids eliminates the source of contamination that would otherwise occur during high alcohol concentration binding, allowing efficient small RNA recovery without protein interference.
2Object-generated harmful factors
If phenol/chloroform extraction is used to remove proteins prior to RNA binding, then protein removal is achieved, but the procedure becomes more complex and toxic
Solution Approach 1:
The patent changes the chemical parameters of protein precipitation by using metal cations (such as zinc chloride or copper sulfate) instead of phenol/chloroform. This parameter change simplifies the procedure by eliminating organic solvent handling while maintaining effective protein removal, and also reduces toxicity without increasing procedural complexity.
3Quantity of substance
If standard RNA isolation procedures are used, then total RNA is isolated, but small RNA is not effectively bound and yield is insufficient
Solution Approach 1:
The patent applies local quality by optimizing binding conditions specifically for small RNA recovery. By using high alcohol concentration (≥45-50%) during the binding step and performing preliminary protein removal, the method creates locally optimized conditions that favor small RNA binding to the solid phase, thereby significantly improving small RNA yield while maintaining total RNA isolation capability.
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 provides high yields of small and large RNA with reduced risk of contamination and toxicity, maintaining RNA integrity and flexibility for downstream analyses, including PCR, without the need for phenol or organic solvents.
Implementation Method 1
adding a precipitation buffer to a sample to prepare an acidic precipitation mixture wherein said precipitation buffer comprises a metal cation precipitant and a buffering agent, has a pH value of 4.0 or less and does not comprise an organic solvent selected from aprotic polar solvents and protic solvents and wherein the acidic precipitation mixture comprises the metal cation precipitant in a concentration of less than 200 mM and precipitating proteins
Data Source
AI summary
A phenol-free method for isolating a nucleic acid from a sample is provided, said method comprising the following steps: a) adding a precipitation buffer to a sample to prepare an acidic precipitation mixture wherein said precipitation buffer comprises a metal cation precipitant and a buffering agent, has a pH value of 4.0 or less and does not comprise an organic solvent selected from aprotic polar solvents and protic solvents and wherein the acidic precipitation mixture comprises the metal cation precipitant in a concentration of less than 200 mM and precipitating proteins; b) separating the precipitate from the supernatant, wherein the supernatant comprises small RNA having a length of less than 200 nt and large RNA having a length of at least 1000 nt; and c) isolating a nucleic acid from the supernatant. The present method allows to avoid the use of organic solvents during protein precipitation. Also provided is a precipitation buffer.