MicroRNA Detection Using Universal Primers and Poly-A Tailing
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Solution Overview
Problem
Current methods for detecting microRNAs are cumbersome, costly, and not suitable for high-throughput analysis due to complex probe and primer design, high sample requirements, and issues with sensitivity and specificity.
Innovation Solution
A method involving a poly-adenylic acid tailing reaction followed by a one-step reverse transcription and PCR amplification using universal primers and fluorescent probes, allowing for simultaneous processing of multiple microRNAs with improved sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If stem-loop primers or miRNA tailing methods are used for reverse transcription amplification detection, then microRNA can be identified and synthesized, but the operation steps become complicated and sensitivity is not high
Solution Approach 1:
The patent combines the tailing reaction system and reverse transcription reaction system into a single one-step reaction system. The reaction mixture includes poly(A) polymerase for tailing, reverse transcriptase for reverse transcription, and a universal primer with poly(T) sequence, allowing both tailing and reverse transcription to occur simultaneously in one reaction vessel, thereby simplifying操作步骤 while maintaining detection sensitivity
Solution Approach 2:
The patent employs a universal primer containing a poly(T) sequence that can bind to the poly(A) tail added to any microRNA molecule. This universal primer serves multiple functions: it acts as a binding site for reverse transcription, provides a common sequence for subsequent PCR amplification, and enables detection of different microRNAs without requiring microRNA-specific primers for each target
2Measurement precision
If custom probes are synthesized for each independent microRNA in PCR process, then specific detection can be achieved, but cost increases and high-throughput detection becomes difficult
Solution Approach 1:
The patent uses a universal fluorescent probe that contains a sequence complementary to the universal primer's extend tag region. This single universal probe can detect all microRNAs that have been reverse transcribed using the universal primer, eliminating the need to synthesize custom probes for each microRNA target while maintaining detection specificity through the probe's complementary binding to the amplified product
Solution Approach 2:
The detection system separates the specificity function into two parts: the forward primer provides microRNA-specific recognition through its sequence complementary to the extended microRNA fragment, while the reverse primer and fluorescent probe provide universal recognition of the universal primer sequence. This segmentation allows one universal probe to work with multiple microRNA-specific forward primers
3Measurement precision
If one reverse transcription process targets a single microRNA using stem-loop primers, then specificity is achieved, but large sample volume is required and high-throughput detection is difficult
Solution Approach 1:
The patent employs a universal reverse transcription primer containing a poly(T) sequence that can bind to the poly(A) tail added to any microRNA molecule. This single universal primer enables simultaneous reverse transcription of multiple different microRNAs in one reaction, reducing the sample volume required per microRNA while maintaining detection specificity through the combination of the forward primer's specific recognition and the probe's specific binding
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 enables efficient detection of microRNAs with reduced sample volume, high sensitivity, and cost-effectiveness, facilitating high-throughput analysis and overcoming the limitations of existing techniques.
Implementation Method 1
adding a poly-adenylic acid tailing reaction system to obtain an RNA molecule with polyadenylic acids at the 3' end; the tailing reaction system contains: an enzyme for catalyzing the poly-adenylic acid tailing reaction
Implementation Method 2
adding a reverse transcription reaction system to obtain a reverse transcribed cDNA product; the catalytic system contains: an enzyme for catalyzing a reverse transcription reaction
Implementation Method 3
amplifying the obtained cDNA reverse transcription product in a DNA amplification reaction and fluorescence detection system
Implementation Method 4
detecting the signal of the reporter group in the cyclic amplification step of the polymerase chain reaction
Data Source
AI summary
Provided is a method for detecting a microRNA. The method comprises: adding both (a) a polyadenylic acid tailing reaction system and (b) a reverse transcription reaction system to a sample to be detected, and subjecting the mixture to a one-step method by means of using a universal reverse transcription primer to obtain a reverse-transcribed cDNA product; and amplifying the product with a downstream universal primer and a specific upstream primer and detecting the amplified product with a universal fluorescence probe in a DNA amplification reaction and fluorescence detection system. Further provided is a kit based on the above-mentioned method. The provided method and kit have significantly higher sensitivity and specificity than traditional methods, can realize high-throughput sensitive detection with a small amount of samples, are convenient to operate and consume a short amount of time, and can detect batches of target microRNAs at low cost. Therefore, the provided method and kit are suitable for the screening of various biological samples, early diagnosis, companion diagnosis and prognosis evaluation of clinical diseases, etc.