Micro ribonucleic acid detection method based on microfluidic bead array chip and DNA polymerase-mediated primer extension technology

A micro ribonucleic acid and microbead array chip technology, which is applied in the field of biological detection, can solve the problems of high throughput and achieve high sensitivity

Inactive Publication Date: 2014-06-04
HUNAN INSTITUTE OF ENGINEERING
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Problems solved by technology

The development of this technology has better solved the problem of high-throughput and high-sensitivity detection under the condition of micro-sample. At the same time, the detection process is simple and does not require expensive detection instruments. It provides a new direction for the development of future automated high-performance biomolecular detection platform technology. , this technique has not been reported for the analysis of microRNA

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  • Micro ribonucleic acid detection method based on microfluidic bead array chip and DNA polymerase-mediated primer extension technology
  • Micro ribonucleic acid detection method based on microfluidic bead array chip and DNA polymerase-mediated primer extension technology
  • Micro ribonucleic acid detection method based on microfluidic bead array chip and DNA polymerase-mediated primer extension technology

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Embodiment

[0025] (1) Preparation of functionalized polystyrene microbeads for microRNA detection: 10-30 μm avidin-modified polystyrene microspheres are used as the immobilized solid-phase interface for microRNA capture probes, and 100 μL of mass is taken The avidin-modified microspheres with a concentration of 1%-3% were placed in a centrifuge tube, and washed with 100 μL affinity eluent (20 mM Tris pH7.5, 1 M NaCl, 1 mM EDTA, 0.0005% wt Triton X-100) for two washes. Once, the centrifugation condition was 3500rpm, 5min, and the supernatant was removed; then 44μL of affinity eluent, 3μL of 0.1-0.3μM microRNA capture probes as shown in Table 1 were added, and incubated at room temperature for 15-30min; The wash method removes unbound molecules and suspends the functionalized microspheres in 100 μL of affinity eluent. The capture probe is specifically combined with the avidin on the surface of the microsphere through the modified biotin and fixed on the surface of the microsphere, thereby ...

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Abstract

The invention discloses a micro ribonucleic acid detection method based on a microfluidic bead array chip and a DNA polymerase-mediated primer extension technology. According to the detection method, a micro ribonucleic acid capture probe modified by biotin is fixed to the surface of an avidin-modified microsphere through a biotin-avidin bonding method to form a functionalized microsphere; the functionalized microsphere is fixed in a micro chamber corresponding to a detection zone of a microfluidic chip so as to form the microfluidic bead array detection chip; then a sample solution containing micro ribonucleic acid is allowed to flow into the microfluidic bead array detection chip, then to flow into a DNA polymerase solution and a biotinylation extension substrate, and finally to flow into a quantum dot labeled by the avidin; and surface fluorescence of the microsphere after the micro ribonucleic acid flows into is calculated to perform qualitative operation and quantification. The method can detect the micro ribonucleic acid with high sensitivity and high specificity and achieves quantification analysis of specificity and sensitivity of the micro ribonucleic acid in a total RNA sample.

Description

technical field [0001] The invention relates to the technical field of biological detection of a micro-total analysis system, in particular to a microRNA detection method based on a microfluidic bead array chip and a DNA polymerase-mediated primer extension technology. Background technique [0002] MicroRNA (miRNA) is a small endogenous non-coding RNA molecule, consisting of about 21-25 nucleotides, usually targeting one or more mRNAs, and inhibiting or breaking target mRNAs at the translational level. Regulates gene expression. More than 3,000 miRNAs have been discovered so far, most of which play a key regulatory role in animals and are one of the most important gene expression regulators. It is estimated that about 2 / 3 of the genes in the human body are regulated by a certain gene. Or the regulation of a set of miRNAs. Therefore, the development of new detection methods for miRNA is of great significance for medical clinical diagnosis. The developed miRNA detection met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6837
Inventor 张何傅昕刘媛朱振军
Owner HUNAN INSTITUTE OF ENGINEERING
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