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MiRNA delivery system verification method

A verification method and system technology, applied in recombinant DNA technology, other methods of inserting foreign genetic materials, etc., can solve the problems of the efficiency of the delivery system and the lack of accurate verification methods for characterization

Pending Publication Date: 2020-09-01
江苏申基生物科技有限公司
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Problems solved by technology

However, due to the effect of negative charge, it is difficult for artificially synthesized miRNA analogs and miRNA inhibitors to pass through the cell membrane and enter the interior of the cell. There is no accurate verification method for the efficiency and characterization of the miRNA delivery system. Based on the previous research of the miRNA delivery tool in our research group, we designed a reduction-responsive miRNA delivery system verification method

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Embodiment Construction

[0042] The present invention will be further described in detail below in conjunction with specific embodiments.

[0043] 1. Examples of specific verification steps and methods

[0044] (1) Agarose gel electrophoresis

[0045] At room temperature, take 100 pmol of miR-34a and azide-modified carbon dots at a mass ratio of 10:1, 20:1, 30:1 and 40:1 for Cu(I)-catalyzed Click reaction for 3 hours, The reaction solution was electrophoresed on 2% ethidium bromide-stained agarose gel, and 100 pmol of free miR-34a was used as a control.

[0046] (2) Cell culture

[0047] The culture conditions of HeLa cells were: high glucose DMEM with 10% fetal bovine serum and 1% penicillin / streptomycin, and the cells were cultured in an incubator containing 5% CO2 at 37°C.

[0048] (3) Control release verification

[0049] The carbon dots loaded with 100 pmol miR-34a were dissolved in PBS containing 10 mM or 2 μM GSH, and the reaction solution was dissolved in 2% bromine at 0 h, 1 h, 2 h, 4 h, ...

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Abstract

The invention relates to a miRNA delivery system verification method. The method comprises following steps: carrying out a biological activity test reaction on acetylene bond modified double-strandedmiR-34a, carrying out a release characterization test reaction on the miR-34a in-vitro in a reducing environment after the miR-34a is successfully loaded on the surface of a carbon dot; respectively testing release data of miR-34a in different GSH environments according to the fact that the concentration of reducing species in cancer cells is higher than that of reducing species outside the cells;carrying out a cell experiment to test the efficiency data of transporting miRNA into cells; secondly, testing transportation and controllable release data of a transportation system on miR-34a in cancer cells, finally verifying the data of miR-34a that is transported into the cells and plays a normal function; and finally, obtaining a verification result of the reduction response type miRNA transport system based on the carbon dots. A miRNA carrying system verification method, which is reasonable in design, clear in verification means and high in result accuracy, is obtained.

Description

technical field [0001] The invention relates to the technical field of calibration products, in particular to a method for verifying an miRNA delivery system. Background technique [0002] Synthetic miRNA analogs (miRNA mimics) and miRNA inhibitors (miRNA inhibitor) have been proven to be effective bioactive macromolecules that regulate miRNA levels in organisms, and are increasingly used in the artificial regulation of miRNA and clinical research. However, due to the effect of negative charge, it is difficult for artificially synthesized miRNA analogs and miRNA inhibitors to pass through the cell membrane and enter the interior of the cell. There is no accurate verification method for the efficiency and characterization of the miRNA delivery system. Based on the previous research of the miRNA delivery tool in our research group, we designed a reduction-responsive miRNA delivery system verification method. Contents of the invention [0003] The purpose of the present inve...

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Application Information

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IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 童坤肖潇黄磊
Owner 江苏申基生物科技有限公司
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