Stepwise Alcohol Addition for High-Yield RNA Isolation
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Solution Overview
Problem
Current RNA isolation methods, especially those without phenol-based extraction, face challenges in achieving high yields of total RNA and small RNA, particularly from fibrous tissues, due to the use of high alcohol concentrations which can reduce RNA recovery and binding efficiency.
Innovation Solution
A step-wise addition of alcohol during the RNA isolation process, combined with a chaotropic agent and proteolytic digest, allows for improved binding conditions, significantly increasing the yield of both total and small RNA without the need for phenol-based extraction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If high alcohol concentration is used for RNA binding to solid phase, then small RNA binding efficiency is improved, but total RNA yield is reduced
Solution Approach 1:
The patent segments the alcohol addition process into multiple steps: first adding alcohol to reach 30-40% concentration for initial binding, then incrementally adding more alcohol to reach final concentration of 50-70%. This stepwise approach allows RNA to bind progressively as alcohol concentration increases, preventing the yield loss that occurs when high alcohol concentration is applied all at once.
Solution Approach 2:
The patent performs preliminary binding at lower alcohol concentration (30-40%) before increasing to the final high concentration (50-70%). This preliminary action allows RNA to begin binding under gentler conditions, ensuring that subsequent increases in alcohol concentration do not cause already-bound RNA to be lost.
2Quantity of substance
If phenol-based extraction is used, then RNA isolation yield is improved, but toxicity and safety issues worsen
Solution Approach 1:
The patent changes the chemical parameters of the extraction system by replacing phenol with phenol-free alternative reagents. The method achieves comparable RNA isolation yields by optimizing alcohol concentration parameters (30-70% range) and using chaotropic salts, thereby eliminating phenol's harmful effects while maintaining effective RNA binding and isolation.
Solution Approach 2:
The patent replaces expensive and hazardous phenol-based reagents with safer, more accessible alcohol-based reagents. The alcohol-based system achieves equivalent or superior results while being less toxic, easier to handle, and eliminating the need for special phenol disposal procedures.
3Productivity
If standard RNA isolation procedures are used, then isolation speed is improved, but small RNA capture efficiency worsens
Solution Approach 1:
The patent makes the alcohol concentration dynamic rather than static. Instead of using a fixed alcohol concentration, the method dynamically adjusts alcohol concentration from 30-40% to 50-70% in steps, allowing the binding conditions to adapt and optimize for capturing small RNAs while maintaining overall process efficiency.
Data Source
AI summary
The present invention provides a method for isolating RNA including small RNA having a size of 200 nt or less from a sample, comprising the following steps: a) providing a composition comprising RNA and a chaotropic agent; b) adding alcohol; c) incubating the mixture for at least 2 min; d) adding additional alcohol to the mixture to adjust the overall alcohol concentration in the mixture to ≥50%; e) binding RNA contained in the mixture to a nucleic acid binding solid phase; f) optionally washing the bound RNA; g) optionally eluting RNA from the solid phase. Due to the step-wise addition of alcohol, the overall RNA yield and the yield of small RNA is improved.