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A method, probe set and kit for detecting miRNAs-21

A miRNA-21, kit technology, applied in the field of molecular biology, can solve the problems of high sequence similarity, low stability, photobleaching, etc., and achieve the effect of high sensitivity and selection

Active Publication Date: 2020-03-31
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current fluorescent sensors have some specific limitations, including small probe size, low stability, photobleaching, spectral overlap with background fluorescence, low abundance and high sequence similarity, etc.

Method used

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  • A method, probe set and kit for detecting miRNAs-21
  • A method, probe set and kit for detecting miRNAs-21
  • A method, probe set and kit for detecting miRNAs-21

Examples

Experimental program
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Effect test

preparation example Construction

[0035] The preparation method of the polydiacetylene microtube of the present invention is specifically as follows: take 0.0056g of aminodiacetylene and 0.00075g of octadecylamine-substituted melamine and dissolve it in 2mL of absolute ethanol solution, and pour the solution into a 300mL ultra- In pure water, sonicate for 60 minutes, place in a dark place to cool naturally to room temperature, and then put in a refrigerator at 4°C overnight to obtain complex vesicles. Add 10ml of vesicle solution and 10μL of lead nitrate solution to the weighing bottle, open it at room temperature and place it in a ventilated place. After two to three weeks, white filamentous substances will precipitate, and polydiacetylene microtubes will be obtained.

[0036] In some embodiments, the preparation method of the polydiacetylene microtube modified by single-stranded DNA of the present invention is specifically: the polydiacetylene microtube is taken out from the weighing bottle, placed under a 25...

Embodiment 1

[0053] Example 1: Preparation of single-stranded DNA modified microtubes

[0054] Dilute 20 μL of allyl glycidyl ether to 10 mL, add polydiacetylene microtubes, and react overnight at 30° C. to obtain double bond-modified polydiacetylene microtubes. Combine a single polydiacetylene microtube with 200 μL of 5'-end thiol-modified single-stranded DNA (ssDNA1 sequence shown in SEQ ID NO: 1), 2959 photoinitiator (n(2959):n(ssDNA1)=1: 100) Mix evenly and react under 365nm light for 6 hours to obtain single-stranded DNA modified polydiacetylene microtubes. The microtube infrared spectrum of single-stranded DNA modified polydiacetylene microtubes is shown in figure 2 b, Raman spectrum of polydiacetylene microtubes modified with single-stranded DNA see figure 2 c.

Embodiment 2

[0055] Example 2: Single-stranded DNA modified gold nanorods

[0056] Gold nanorods were prepared by the seed growth method, and after purification, they were incubated with 5'-end thiol-modified single-stranded DNA (ssDNA2 sequence shown in SEQ ID NO: 2) (the molar ratio of gold nanorods to DNA was 1:25600) , to obtain single-stranded DNA modified gold nanorods. The specific operation is as follows: first, add 250 μL of 0.01 M chloroauric acid to 7.5 mL of 0.1 M cetyltrimethylammonium bromide (CTAB) solution, and stir evenly. Then 600 μL of 0.01 M sodium borohydride solution was added (sodium borohydride was placed at -20° C. for 10 minutes in advance), and then stirred for 2 minutes under mechanical stirring (400 rpm). After 2 minutes, a light brown solution will form, which is the gold seed solution. The gold seed solution should be left at room temperature for 1 hour before use. Preparation of growth solution: under mechanical stirring conditions (stirring speed is 250r...

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Abstract

The present invention belongs to the field of molecular biology, and specifically discloses a method, a primer group and a kit for detecting miRNAs-21. According to the present invention, in the method, gold nano-rod modified single-stranded DNA and polydiacetylene micro-tube modified single-stranded DNA form gold nano-rod modified polydiacetylene micro-tubes through hybridization pairing so as to quench the polydiacetylene micro-tube end head optical waveguide fluorescence, the miRNA-21 can catalyze the self-assembly of the hairpin probe to form the double-stranded structure after the gold nano-rod modified polydiacetylene micro-tubes, the H1 probe, the H2 probe and the sample to be tested are mixed, and single-stranded substitution is performed through toehold so as to recover the polydiacetylene micro-tube end head optical waveguide fluorescence, such that the miRNAs-21 content in the sample to be tested can be detected according to the fluorescence intensity change; and the experiment results show that the detection method has advantages of high miRNAs-21 detection sensitivity, high miRNAs-21 selectivity, simple, sensitive and fast detection process, and accurate detection result.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting miRNAs-21, a probe set and a kit. Background technique [0002] MicroRNAs (miRNAs) are a class of non-coding single-stranded small RNA molecules composed of 18-24 bases, which negatively regulate gene expression at the post-transcriptional level and participate in important biological processes such as cell growth, development, and apoptosis. Some recent medical studies have shown that the expression level of miRNA, either increased or decreased, is inextricably linked to major human diseases such as cancer to some extent. This allows miRNAs to be used as tumor markers to monitor cancer at an early stage. [0003] The coding gene of miRNA-21 is located at 17q23.2, which is the coding region of vacuole membrane protein-1 (VMP1), the tenth intron of VMP1 gene. VMP1 is also known as transmembrane protein-49 (transmembraneprotein-49, TMEM-49). miRNA-21 is hi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6825C12Q1/6886C12N15/11
CPCC12Q1/6825C12Q1/6886C12Q2600/178C12Q2563/107
Inventor 邹纲王梦乔
Owner UNIV OF SCI & TECH OF CHINA
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