A method for isolating exosomes from animal plasma and performing purity testing

A technology of purity detection and exosomes, which is applied in the field of cell biology to achieve high biological activity

Inactive Publication Date: 2020-02-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the identification of exosome purity requires the use of some expensive instruments such as electron microscopes, this is a great limitation for many laboratories

Method used

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  • A method for isolating exosomes from animal plasma and performing purity testing
  • A method for isolating exosomes from animal plasma and performing purity testing
  • A method for isolating exosomes from animal plasma and performing purity testing

Examples

Experimental program
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Effect test

Embodiment 1

[0047] A 2.7Kg rabbit was collected 10ml of blood by way of jugular vein blood collection, and the anticoagulant was EDTA. The anticoagulated blood was transferred into a 2ml centrifuge tube and centrifuged at 3000rpm for 30min to separate the plasma. The separated plasma was used for exosome extraction.

[0048] Methods for isolating exosomes from rabbit plasma, such as figure 1 shown, including the following steps:

[0049] (1) Dilute 2ml of plasma with PBS solution at a ratio of 1:1;

[0050] (2) Centrifuge at 2000g for 30min at 4°C to remove cellular components in the plasma, and retain the supernatant after centrifugation;

[0051] (3) Collect the filtrate into a centrifuge tube again, and centrifuge at 12,000 g for 30 minutes at 4°C to remove cell debris. If the plasma needs to be frozen, store it at -80°C after this step;

[0052] (4) Collect the supernatant into a centrifuge tube, filter the supernatant with a 0.22 μm filter membrane, so that microcapsules smaller ...

Embodiment 2

[0061] The exosomes isolated in Example 1 were subjected to total RNA extraction, miRNA quantification and purity detection, and the miRNA extracted in Example 1 was subjected to poly(A) reverse transcription and quantification.

[0062] The present invention utilizes 6 highly expressed miRNAs identified in human plasma for miRNA quantitative detection, namely miR-93a-3p, miR-451a-5p, miR-21-5p, miR-16-5P, miR-181 -5p, miR-92a; two additional intracellular genes were used: U6 and 5S. U6 is a small nuclear molecule RNA, which mainly exists in the nucleus. 5S rRNA is a component of the large ribosomal subunit. Both prokaryotes and eukaryotes have 5S rRNA, and their structures are similar. Both types of genes are not present in exosomes and thus serve as negative controls. The primer sequences of the 12 genes are shown in the list. Specific steps are as follows:

[0063] Poly(A) tailing reaction reverse transcribes miRNA into cDNA. Specific steps are as follows:

[0064] (1...

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Abstract

The invention provides a method for separating exosomes from animal plasma and for detecting purity. The method mainly comprises the following steps: first, thinning the plasma by virtue of a PBS (phosphate buffer solution) at the ratio of 1 to 1; removing cell components and cell debris in the plasma through centrifuging; precipitating the exosomes through ultrahigh-speed centrifugation; suspending the exosomes by virtue of 250[mu]l of the PBS solution; extracting total RNA in an exosome suspension by virtue of Trizol LS; and conducting poly (A) reversing and related gene quantification on the extracted total RNA, and detecting the purity. The method disclosed by the invention is conducive to researches on plasma exosome contents and exosome related functions in the future.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for isolating exosomes from animal plasma and performing purity detection. Background technique [0002] Exosomes are membranous vesicles with a diameter of about 50-200 nm. Cells produce small vesicles through endocytosis, and the small vesicles fuse to form early endosomes, which gradually become late endosomes. With the entry of some "cargo" such as miRNA, enzyme molecules, and heat shock proteins in the cytoplasm, many small vesicles (intraluminal vesicles, ILVs) will be produced in the late endosome, and gradually evolve into multivesicular bodies (MVB); These vesicles are then released outside the cell to form exosomes. Exosomes exist in cell culture fluid and body fluids, mainly including saliva, pleural fluid, cerebrospinal fluid, ascites, urine, plasma, serum and other body fluids. Exosomes contain a variety of biomolecules, such as mRNA, miRNA, mtDNA,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6806
CPCC12Q1/6806C12Q1/6851C12Q2563/107C12Q2545/113C12Q2565/113
Inventor 龙科任马继登李明洲李学伟顾以韧
Owner SICHUAN AGRI UNIV
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