Method for separating exosomes from animal plasma and for detecting purity

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

Inactive Publication Date: 2016-07-20
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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  • Method for separating exosomes from animal plasma and for detecting purity
  • Method for separating exosomes from animal plasma and for detecting purity
  • Method for separating exosomes from animal plasma and for detecting purity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[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 12,000g for 30 minutes at 4°C to remove cell debris. If the plasma needs to be cryopreserved, it should be stored at -80°C after this step;

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

Embodiment 2

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

[0062] The present invention utilizes 6 high-expressed miRNAs identified in human plasma for quantitative detection of miRNAs, 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 RNA, which mainly exists in the nucleus. 5SrRNA is a component of the large ribosomal subunit, both prokaryotes and eukaryotes have 5SrRNA, and the structure is similar. Neither of these two types of genes were present in exosomes, so they could be used as negative controls. Primer sequences for 12 genes are shown in the table. Specific steps are as follows:

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

[0064] (1) Poly(A) tail...

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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, in particular to a method for separating exosomes from animal plasma and performing purity detection. Background technique [0002] Exosomes are membranous vesicles with a diameter of about 50-200 nm. Cells generate small vesicles through endocytosis, and the small vesicles fuse to form early endosomes (earlyendosomes), which gradually become lateendosomes. With the entry of some "cargoes" such as miRNA, enzyme molecules, and heat shock proteins in the cytoplasm, the late endosome produces many intraluminalvesicles (ILVs), which gradually evolve into multivesicular bodies (MVB); then , these small vesicles are released extracellularly to form exosomes. Exosomes exist in cell culture fluids 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, proteins,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
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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