Artificial liposome containing miRNA mimic and preparation method and application thereof
A technology of liposomes and artificial lipids, which is applied in biochemical equipment and methods, microbiological determination/testing, etc. It can solve the problems that are difficult to normalize with markers, interference factors cannot be ruled out, and clinical sample capsules cannot be effectively used. Bubble miRNA abundance analysis and other issues
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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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