RT-PCR Reagent Solution for Direct RNA Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Traditional reverse transcription polymerase chain reaction (RT-PCR) methods require RNA extraction from biological samples, which is labor-intensive, costly, and prone to contamination, and lacks effective inhibitors for direct RNA amplification from crude samples like blood.
Innovation Solution
A method and composition for RT-PCR that uses a solution comprising a polar aprotic solvent, serum albumin, and a polyol, along with optional reducing agents, non-ionic surfactants, and betaines, allowing direct amplification of RNA from crude biological samples without prior extraction, using a dried reagent composition that includes a sequestering agent, polymerase, and deoxyribonucleotide triphosphates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA extraction is performed prior to RT-PCR amplification, then RT-PCR inhibitors are removed from the sample, but the process becomes labor-intensive, costly, and prone to cross-contamination
Solution Approach 1:
The invention extracts and removes RT-PCR inhibitors directly from crude biological samples using a specialized solution containing polar aprotic solvent, serum albumin, and polyol, eliminating the need for separate RNA extraction steps while maintaining inhibitor removal effectiveness
Solution Approach 2:
The invention combines inhibitor removal, reverse transcription, and PCR amplification into a single integrated reaction system, where the solution with polar aprotic solvent, serum albumin, and polyol performs multiple functions simultaneously, reducing procedural steps and contamination risks
2Reliability
If RNA extraction is performed prior to RT-PCR amplification, then the risk of cross-contamination is reduced, but the process becomes more complex and time-consuming
Solution Approach 1:
The invention merges inhibitor removal, reverse transcription, and PCR amplification into a single integrated reaction system, reducing the number of separate processing steps and minimizing opportunities for cross-contamination while maintaining reliability
Solution Approach 2:
The solution containing polar aprotic solvent, serum albumin, and polyol acts as an intermediary that directly treats crude samples in the reaction vessel, eliminating the need for intermediate extraction steps and reducing contamination risks between steps
3Productivity
If direct RT-PCR is performed from crude samples without extraction, then labor and time are saved, but RT-PCR inhibitors in the sample prevent effective amplification
Solution Approach 1:
The invention converts the harmful effect of RT-PCR inhibitors into a beneficial outcome by using serum albumin to bind and sequester inhibitors like heme, transforming the crude sample's problematic components into non-interfering complexes that allow successful amplification
Solution Approach 2:
The invention changes the chemical parameters of the reaction environment by introducing polar aprotic solvent and polyol, which alter the solubility and interaction properties of inhibitors, enabling effective amplification from crude samples without extraction
4Reliability
If inhibitor-resistant DNA polymerases are used for direct PCR, then amplification from crude samples is improved, but no alternatives exist for direct RT-PCR amplification of RNA
Solution Approach 1:
The invention creates a composite reaction system combining polar aprotic solvent, serum albumin, polyol, reverse transcriptase, and DNA polymerase, where each component contributes specific properties that collectively enable inhibitor-resistant RT-PCR amplification from crude samples
Solution Approach 2:
The solution containing polar aprotic solvent, serum albumin, and polyol serves as an intermediary system that protects and enables the reverse transcriptase and DNA polymerase to function effectively in the presence of inhibitors, filling the gap for inhibitor-resistant RT-PCR reagents
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
Enables efficient and reliable direct RT-PCR from crude biological samples, reducing contamination risks and labor, while overcoming inhibitor challenges, thereby improving the robustness and efficiency of RNA amplification.
Implementation Method 1
the RT-PCR is carried out in a solution comprising a polar aprotic solvent, a serum albumin, and a polyol
Implementation Method 2
a DNA molecule (termed a complementary DNA molecule, which can be abbreviated to a 'cDNA molecule') is generated from a single stranded RNA template using a reverse transcriptase
Implementation Method 3
The cDNA is then used as a template for exponential amplification using PCR
Data Source
AI summary
The present invention provides a method of amplifying an RNA molecule in a biological sample by reverse transcription PCR (RT-PCR), wherein the RT-PCR is carried out in a solution comprising a polar aprotic solvent; a serum albumin, and a polyol.