Plasma microRNA detection kit and detection method based on allglo probe fluorescence quantitative PCR

Abstract

The invention discloses a plasma microRNA (Ribonucleic Acid) detection kit and detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction), relating to microRNA. The kit is provided with a kit body, a partition plate, a miRNA extraction reagent bottle, a stem-loop reverse transcription reagent bottle and a real-time fluorescent quantitative PCR reagent bottle. The detecting method comprises the steps: after sufficiently cracking 200mu L of plasma, adding the plasma with the working concentration of 5n mol as a microRNA exogenous reference, immediately carrying out vortex vibration for 15s, and carrying out the rest steps according to the conventional method, wherein the plasma with the working concentration of 5n is contained in the kit; carrying out reverse transcription on miRNA by using a stem-loop reverse transcription reagent provided by the kit to form cDNA; carrying out real-time fluorescent quantitative PCR amplification on cDNA by using a real-time fluorescent quantitative PCR reagent, wherein the real-time fluorescent quantitative PCR reagent is provided by the kit; and comprehensively analyzing all data provided by instruments, setting reasonable threshold values and base lines and finally carrying out analysis. The detection kit is high in specificity and sensibility and capable of rapidly and accurately detecting the content of miRNA in a sample.

Description

technical field [0001] The invention relates to microRNA, in particular to a plasma microRNA detection kit based on AllGlo probe fluorescent quantitative PCR and a detection method thereof. Background technique [0002] MicroRNA (miRNA) is a kind of non-coding RNA molecule of about 22 nucleotides widely present in eukaryotic cells, which participates in many physiological and pathological processes in organisms, by regulating gene expression, in cell proliferation, apoptosis , growth and development, cell differentiation, metabolism and other processes play an important role. The specific performance is that miRNA forms the initial primary transcript pri-miRNA to pre-miRNA in the nucleus, and then transports out of the nucleus, forms mature miRNA by shearing, and forms RISC (RNA-induced silencing complex) with Ago protein, etc. Partially inhibit or degrade the target mRNA sequence and participate in the regulation of gene expression (BartelDP. MicroRNAs: genomics, biogenesi...

AI Technical Summary

Problems solved by technology

Among them, northern blot technology is one of the early means of RNA detection, but its specificity and sensitivity are low. If the sample RNA content is low or there is degradation, it may not be detected; in situ hybridization technology is used to detect the distribution of miRNA at the tissue and cell level , while performing semi-quantitative detection of miRNA, its disadvantage is that it cannot accurately detect low-expression miRNA; microarray chip technology is a high-throughput detection technology that can simultaneously detect a large number of miRNAs in one or more samples , but there are problems such as time-consuming and labor-intensive chip fabrication, high labeling costs, and low detection sensitivity and specificity.

The reverse transcription primer based on the stem-loop structure can specifically recognize and amplify the mature miRNA sequence, but the precursor of the miRNA has not been amplified. The disadvantage of the Taqman-MGB probe for detecting miRNA is that the synthesis is expensive, which is not conducive to popularization.

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