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Artificial liposome containing miRNA mimic and its preparation method and application

A liposome and man-made technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problem of inability to eliminate interference factors, inability to effectively analyze vesicle miRNA abundance in clinical samples, and difficult to use markers to return Unified reference and other issues

Active Publication Date: 2020-06-16
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This external reference method of free miRNA mimic can only exclude the interference factors in the RNA extraction process, but cannot exclude the interference factors in the vesicle isolation stage, and it is difficult to be used as a reference for the normalization of markers between different samples, so it is not effective Abundance analysis of vesicular miRNAs in clinical samples

Method used

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  • Artificial liposome containing miRNA mimic and its preparation method and application
  • Artificial liposome containing miRNA mimic and its preparation method and application
  • Artificial liposome containing miRNA mimic and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Preparation of artificial liposomes (fluorescent liposomes) containing miRNA mimic

[0048] 1. Preparation of liposomes by constant pressure controlled extrusion

[0049] Use a 250 μL sealed glass syringe to add 50 μL chloroform into a 20 mL glass vial; then use a 100 μL sealed glass syringe to add 15 μL methanol to the same glass vial; then add 10 mg / mL phosphatidylcholine 108 μL, 10 mg / mL phosphatidylethanolamine 21 μL and 10 μL of 11 mg / mL cholesterol were sequentially added to the above-mentioned glass bottle; the organic solvents chloroform and methanol were evaporated with nitrogen until the formation of thin film lipid was observed at the bottom of the bottle; the glass bottle was placed in a vacuum desiccator for at least 30 minutes to remove the residual organic solvent; then Add 1 ml of PBS buffer dissolved with miRNA mimic (containing mimic 1: 50 fmol; mimic 2: 500 fmol; mimic 3: 5 pmol) and 20 μL of 11 mg / mL RNase inhibitor into the glass vial. ...

Embodiment 2

[0052] Example 2 Identification of artificial liposomes containing miRNA mimic

[0053] Take 10 μL of liposomes prepared in Example 1, and use ddH 2 O was diluted at a volume ratio of 1:10; the diluted liquid was transferred to the carbon membrane for adsorption for 10 minutes, and then washed with DI water (deionized water), and uranyl acetate UO 2 (CH 3 COO) 2 2H 2 O, after staining for 1 minute, use a transmission electron microscope (TEM, model JEOL-JEM1400) to visually detect the shape of the liposomes, and take pictures for recording. The result shows that the particle size of gained liposome is 30-150nm ( figure 1 , a).

[0054] Take 10 μL of the liposome prepared in Example 1, add 700 μL of QIAzol reagent, use the column adsorption method for RNA extraction, dissolve the RNA in 30 μL of RNase-free water, and perform PCR detection.

[0055] The primer information is as follows:

[0056]

[0057] The reverse transcription reaction system is as follows:

[0058...

Embodiment 3

[0069] Example 3 Stability analysis of artificial liposomes containing miRNA mimic

[0070] The prepared artificial liposomes were stored at 4°C, and samples were taken on the 0th, 2nd, 8th, 13th, 34th, and 45th days, and 10 μL was taken each time for RNA extraction and PCR quantitative analysis. Abundance of RNA relative to day 0. The result shows that this liposome RNA has good stability, even if preserve more than 45 days, the CT value variation range when each RNA carries out PCR is within 0.4%, shows good stability ( figure 2 ).

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Abstract

The invention provides an artificial liposome containing miRNA mimic and a preparation method and application thereof. The artificial liposome is prepared by mixing liposome with a lipid bilayer structure and miRNA mimic, wherein the miRNA mimic is a synthetic non-human miRNA and its derivatives. A synthetic liposome wrapped with the exogenous miRNA mimic is used for characterization of miRNA extraction efficiency of extracellular vesicle samples and quantitative analysis of extracellular vesicle miRNA in body fluid samples, thus providing strong technical support for stable quantitative detection of vesicle miRNA in clinical needs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an artificial liposome containing miRNA mimic and its preparation method and application. Background technique [0002] Extracellular Vesicles (EVs) refer to vesicle-like bodies with a double-membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 30-1000 nm. Extracellular vesicles are mainly composed of microvesicles. (MicroVesicles, MVs) and exosomes (exosomes), microvesicles are small vesicles that are shed from the cell membrane after cell activation or injury. Due to the unique biological characteristics of extracellular vesicles, they are of great significance in disease diagnosis, especially exosomes. [0003] Exosomes are a kind of membranous vesicles with a particle size of 30-150 nm secreted into the extracellular environment after the fusion of intracellular multivesicular bodies and cell membranes. It plays an important ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6876C12Q1/6806C12Q1/6851
CPCC12Q1/6806C12Q1/6851C12Q1/6876C12Q2600/158C12Q2600/166C12Q2600/178C12Q2525/207C12Q2531/113C12Q2545/114
Inventor 孔关义赵立波周卫陈亚庆
Owner 北京恩泽康泰生物科技有限公司
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