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Micro ribonucleic acid 122 (miR-122) probe and preparation method thereof

A microRNA and probe technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of microRNA function loss and other problems, and achieve low cost, high specificity, and easy operation Effect

Active Publication Date: 2017-08-18
李金波
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these types of methods, only the third method can directly isolate and identify the target genes of microRNAs with high throughput, but recent studies have also found that direct modification of biotin at the 3' end of microRNAs can cause microRNAs Loss of ribonucleic acid function, which suggests an urgent need to develop a group that can replace biotin for the labeling of microribonucleic acid-122

Method used

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  • Micro ribonucleic acid 122 (miR-122) probe and preparation method thereof
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  • Micro ribonucleic acid 122 (miR-122) probe and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0035] Example 1: Synthesis of small molecules and miR-122 probes

[0036] Step 1: Synthesis of Small Molecules

[0037]

[0038] Dissolve 220 mg or 0.5 mmol of compound 1a in 10 mL of tetrahydrofuran, add 43 mg, 0.5 mmol of compound 1b and 100 μL of DIPEA, respectively, stir overnight, distill under reduced pressure and use directly in the next step.

[0039] Step 2: Synthesis of miR-122 probe

[0040]

[0041] The product obtained in the previous step was dissolved in anhydrous DMSO at a concentration of 5 mM. Amino-modified miR-122 was dissolved (50 μM) in PBS (pH=7.4), and the same volume of DMSO was added to make a 5 mM stock solution. Reacted overnight at room temperature, purified by HPLC and lyophilized to obtain miR-122 probe.

Embodiment example 2

[0042] Example 2: Luciferase experiment:

[0043] Step 1: Construction of reporter plasmid

[0044] The 3'UTR end of the Luciferase reporter plasmid was cut with HindIII and Spel endonucleases, and the resulting fragments were separated and purified by electrophoresis. The complementary sequence of miR-122 was inserted into the purified luciferase reporter plasmid through DNA ligase, T4 ligase, and transformed into competent Escherichia coli, and then screened through a screening plate containing ampicillin to obtain possible positive plasmids. Finally, the reporter plasmid was sequenced to confirm its correctness.

[0045] Step 2: Luciferase Assay Analysis

[0046] The luciferase reporter plasmid with the complementary sequence of miR-122 was transfected into HepG2 cells. First, the HepG2 cells were planted in a 24-well plate. When the cell density reached 70%-80%, 0.25 μg miR-122 The reporter plasmid and 0.15 μg β-galactosidase internal reference plasmid were transfected ...

Embodiment example 3

[0047] Example 3: Determination of miRNA expression

[0048] Take out 5 μL of RNA for reverse transcription reaction to prepare corresponding cDNA samples. The specific reverse transcription reaction 10 μL system is:

[0049] 5x AMV buffer (Takara) 2 μL

[0050] AMV (Takara) 0.5 μL

[0051] RT primer (ABI) 0.5 μL

[0052] dNTP mixture (Takara) 1 μL

[0053] DEPC H2O 1 μL

[0054] RNA 5 μL

[0055]The reaction program is: 16°C for 30min, 42°C for 30min, 85°C for 5min, and store at 4°C. The prepared cDNA was then used for real-time quantification of miR-122 by probe method (Taqman) PCR, 20 μL;

[0056] The Taqman PCR reaction system is:

[0057] 10x buffer (Takara) 2 μL

[0058] MgCl2 (25mM) (Takara) 1.2 μL

[0059] Taq (Takara) 0.3 μL

[0060] dNTP mixture (10mM) 0.4 μL

[0061] Probe (ABI) 0.33 μL

[0062] cDNA 1 μL

[0063] Distilled H2O 14.77 μL

[0064] The PCR reaction program was 95°C for 5 min, 95°C for 15s, 60°C for 1min, and 40 cycles of amplification. R...

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Abstract

The invention relates to a micro ribonucleic acid 122 (miR-122) probe and a preparation method thereof. The probe consists of three parts: i.e., miR-122, a linking group and an azide group suitable for a click chemical reaction; the probe prepared by combining the three parts does not affect the functions of the miR-122 in hepatocytes; after the probe is combined with a target gene, an affinity group is modified by means of the click chemical reaction, so that the enrichment and detection of the target gene of the miR-122 are realized; therefore, an miR-122 regulatory network of the miR-122 in normal hepatocytes and hepatocellular carcinoma cells can be disclosed by means of the analysis, and a new explanation is provided for the molecular mechanism of the miR-122 in a development process of hepatocellular carcinoma. Compared with the traditional biochemical method, the miR-122 probe has the advantages of being high in specificity, simple and convenient to operate, low in cost, suitable for popularization, and the like. The invention is characterized by providing the miR-122 probe, which is convenient for marking, separation and identification of the miR-122 and the downstream target gene thereof, and the preparation method of the miR-122 probe.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a microribonucleic acid 122 probe and a preparation method thereof. Background technique [0002] MicroRNAs are endogenous small RNA molecules that can negatively regulate gene expression at the post-transcriptional level. Studies in recent years have found that the abnormal expression or function of microRNA can cause the disorder of downstream proto-oncogenes or tumor suppressor genes, thereby inducing or promoting the occurrence and development of diseases. Therefore, microRNA is closely related to the occurrence and development of many diseases including cancer, which has greatly promoted the use of microRNA as a biological target for disease diagnosis and treatment, especially in recent years including let-7, microRNA- Some miRNAs, including 34, have successfully entered clinical research, indicating the great potential of microRNAs in clinical applications. MicroRNA-12...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/141
Inventor 李金波童坤
Owner 李金波
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