One-Step RT-PCR Quantification of WT1 mRNA Expression
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Solution Overview
Problem
Existing methods for quantifying the expression level of the WT1 gene are time-consuming and complex, requiring separate measurements for WT1 mRNA and housekeeping genes, which complicates the process.
Innovation Solution
A one-step RT-PCR method that simultaneously subjects human WT1 mRNA and a housekeeping gene mRNA to reverse transcription and extension reactions in the same vessel, using specific primer sets and probes for efficient and sensitive detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If two-step RT-PCR method is used to measure WT1 mRNA and β-actin mRNA separately, then measurement precision is maintained, but measurement time and operational complexity increase significantly
Solution Approach 1:
The patent combines the measurement of WT1 mRNA and β-actin mRNA into a single one-step RT-PCR reaction by incorporating both target-specific primer sets and probe sets into one reaction mixture. This merging of separate measurements into a unified process reduces measurement time while maintaining quantification accuracy through simultaneous amplification and detection of both targets.
Solution Approach 2:
The patent creates a universal one-step RT-PCR system that can simultaneously detect and quantify multiple targets (WT1 mRNA and β-actin mRNA) using a single reaction protocol. This multi-functional approach eliminates the need for separate reaction setups and procedures, thereby reducing time loss while preserving measurement precision through standardized detection methods.
2Measurement precision
If two-step RT-PCR method is used with separate extension reactions, then measurement precision is maintained, but device complexity and operational steps increase
Solution Approach 1:
The patent merges reverse transcription and PCR amplification into a single one-step reaction process, eliminating the need for separate extension reactions. Both WT1-specific and β-actin-specific primer sets are included in the same reaction mixture, reducing operational complexity while maintaining measurement precision through unified reaction conditions and simultaneous detection.
Solution Approach 2:
The patent develops a universal one-step RT-PCR methodology that handles multiple targets (WT1 and β-actin) within a single reaction system. This multi-functional approach simplifies the overall process by using one reaction protocol for all measurements, thereby reducing device complexity and operational steps while preserving quantification accuracy.
3Productivity
If one-step RT-PCR method is used to simultaneously measure WT1 mRNA and housekeeping gene, then measurement time is reduced, but measurement precision may be compromised
Solution Approach 1:
The patent combines WT1 mRNA and housekeeping gene measurement into a single one-step RT-PCR reaction with optimized primer and probe concentrations. By carefully balancing the reaction conditions and incorporating both target-specific primer sets and probe sets in appropriate ratios, the method achieves rapid simultaneous quantification without compromising measurement precision.
Solution Approach 2:
The patent creates a universal one-step RT-PCR system that efficiently handles multiple targets with optimized reaction parameters. The method maintains high productivity through simultaneous amplification and detection, while preserving quantification accuracy through standardized probe-based detection and optimized reaction conditions for multiple targets.
4Measurement precision
If competitive quantification method using β-actin as standard is used, then measurement precision is maintained, but ease of operation deteriorates due to multiple separate measurements
Solution Approach 1:
The patent merges WT1 mRNA quantification and β-actin normalization into a single one-step RT-PCR reaction. This eliminates the need for separate competitive quantification procedures and multiple separate measurements, thereby improving ease of operation while maintaining measurement precision through simultaneous amplification and probe-based detection of both targets in the same reaction.
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 allows for the convenient and rapid quantification of WT1 mRNA expression levels with higher sensitivity than traditional two-step RT-PCR methods, facilitating diagnosis and prognosis of leukemia and solid cancers.
Implementation Method 1
simultaneously subjecting human WT1 mRNA and a housekeeping gene mRNA to reverse transcription and extension reactions
Implementation Method 2
a primer set used for PCR amplification of the housekeeping gene comprises a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 7 or 12
Data Source
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AI summary
A method for quantifying the expression level of human WT1 mRNA conveniently, in a short period of time, and with high sensitivity is provided. The method can be used for diagnosing cancer, such as leukemia and solid cancer, or for determining when to perform bone marrow transplantation. The method is for quantifying the expression level of human WT1 mRNA by one-step RT-PCR and comprises simultaneously subjecting the human WT1 mRNA and a housekeeping gene (mRNA) to reverse transcription and extension reactions carried out sequentially in the same vessel.