Multiplex miRNA Quantification via Universal Primers
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Solution Overview
Problem
Current methods for analyzing microRNAs as biomarkers are labor-intensive and costly due to the need for individual reactions for each miRNA, making them unsuitable for routine clinical tests.
Innovation Solution
A method involving universal reverse transcription followed by two PCR amplifications with specific and universal primers, allowing simultaneous amplification and quantification of multiple miRNAs in a single assay, using tagged primers and a control nucleic acid to maintain concentration correlation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual detection reactions are performed for each cDNA in separate tubes, then measurement precision of each miRNA is improved, but hands-on time and reagent consumption increase significantly
Solution Approach 1:
The patent combines multiple individual detection reactions into a single multiplex PCR reaction by using a universal primer pair that can amplify multiple different cDNA targets simultaneously. Each cDNA is labeled with a unique fluorescent probe, allowing individual quantification within the same reaction tube, thus reducing hands-on time while maintaining measurement precision.
Solution Approach 2:
The patent employs a universal primer pair that serves multiple functions by being able to bind to and amplify multiple different cDNA sequences simultaneously. This universal primer design allows a single PCR reaction to perform what would traditionally require multiple separate reactions, significantly reducing the time and reagents needed.
2Measurement precision
If individual detection reactions are performed for each cDNA in separate tubes, then measurement precision of each miRNA is improved, but reagent consumption increases
Solution Approach 1:
The patent combines multiple individual detection reactions into a single multiplex PCR reaction by using a universal primer pair that can amplify multiple different cDNA targets simultaneously. Each cDNA is labeled with a unique fluorescent probe, allowing individual quantification within the same reaction tube, thus reducing hands-on time while maintaining measurement precision.
Solution Approach 2:
The patent employs a universal primer pair that serves multiple functions by being able to bind to and amplify multiple different cDNA sequences simultaneously. This universal primer design allows a single PCR reaction to perform what would traditionally require multiple separate reactions, significantly reducing the time and reagents needed.
3Productivity
If multiple miRNAs are analyzed simultaneously, then productivity is improved, but device complexity increases
Solution Approach 1:
The patent employs a universal primer pair that serves multiple functions by being able to bind to and amplify multiple different cDNA sequences simultaneously. This universal primer design allows a single PCR reaction to perform what would traditionally require multiple separate reactions, significantly reducing the time and reagents needed.
Solution Approach 2:
The patent uses fluorescently labeled probes specific to each cDNA target, where each probe emits a distinct fluorescent signal. This allows the detection system to differentiate and quantify multiple miRNAs simultaneously based on their unique fluorescent signatures, increasing productivity without requiring separate physical reaction chambers for each target.
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 significantly reduces hands-on time and reagent usage, enabling efficient and accurate quantification of multiple miRNAs with minimal manual intervention while maintaining the correlation between cDNA and miRNA concentrations.
Implementation Method 1
The extracted miRNAs are commonly transcripted into their corresponding cDNAs by reverse transcription
Implementation Method 2
Amplifying each cDNA by performing a first PCR amplification using a pair of tagged primers
Implementation Method 3
each tagged primer comprising a specific moiety and a tagged moiety, one of the specific moiety being designed for hybridizing to a cDNA
Data Source
AI summary
The present invention relates to a method for quantifying a plurality of miRNA, said method comprising the successive step of providing a sample comprising a plurality of cDNA, each cDNA corresponding to a miRNA. Then, the sample contacted with a plurality of pairs of tagged primers and a pair of universal primers, each tagged primer comprising a specific moiety and a tagged moiety. Subsequently, each cDNA is amplified by performing a first PCR amplification using a pair of tagged primers, wherein annealing step is performed at a first annealing temperature T1, to provide a plurality of DNA. Furthermore, each DNA is amplified by performing a second PCR amplification using the pair of universal primers, wherein annealing step is performed at a second annealing temperature T2. Finally, the concentration each cDNA in the sample is determined to deduce the concentration of each miRNA.