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Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof

A technology of exosomes and reagents, which is applied in the field of plasma exosome extraction reagents, can solve the problems of low extraction efficiency, high equipment requirements, loss of exosomes, etc., and achieve low initial sample volume, high extraction efficiency, and low extraction cost. low effect

Pending Publication Date: 2022-06-03
大连博格林生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Disadvantages of the ultracentrifugation method: time-consuming, the entire extraction process takes at least 8-24 hours; the extraction efficiency is low, and it has been reported in the literature that the exosome extraction efficiency of ultracentrifugation is only 10%; equipment requirements are high, and conventional laboratories do not have ultra-speed Centrifugal conditions
[0006] Disadvantages of the magnetic bead precipitation method: high cost, expensive magnetic bead-conjugated antibodies, generally only for micro-samples, not suitable for exosome extraction of large-volume samples; extraction efficiency is low, due to the specificity of magnetic bead-labeled antibodies, Only exosomes that specifically express the corresponding antigen can be extracted, which limits the extraction effect of this method;
Disadvantages of ultrafiltration: This method usually needs to be combined with ultracentrifugation, and exosomes may be lost due to the adhesion of the filter membrane, and the pressure and shear force during filtration may damage the deformation of exosomes

Method used

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  • Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof
  • Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof
  • Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Mouse plasma exosome extraction and related detection

[0044] 1. Preparation of Plasma

[0045] 1) Add a certain proportion of anticoagulant to the blood collection tube in advance. After collecting blood, slowly invert the blood collection tube and mix well, and let the mixed whole blood stand at room temperature for 1 hour or 2-8 °C overnight;

[0046] 2) Centrifuge at 3000xg at 4°C for 5-10 minutes. The transparent yellow liquid in the upper layer is the plasma. Carefully transfer the supernatant to a new centrifuge tube. Be careful not to aspirate the red blood cells below.

[0047] 3) The collected plasma can be directly used for subsequent exosome extraction experiments or stored in a -80°C refrigerator after being subpackaged.

[0048] 2. Prepare the sample

[0049] 1) Place the plasma (fresh samples or frozen samples thawed in a 25°C water bath) on ice;

[0050] 2) Centrifuge at 2000xg for 20 minutes at 4°C to remove residual cells and debris;

[0051] 3) T...

Embodiment 2

[0089] Extraction of rat plasma exosomes and related detection

[0090] 1. The operation methods of sample processing, exosome extraction and resuspension are the same as those in Example 1.

[0091] Figure 4 The results show that in the case of the same sample amount, the amount of exosome precipitate extracted by the present invention is no different from that of the imported control.

[0092] 2. Comparison of the total amount of RNA extracted from plasma exosomes (rat).

[0093] The methods of sample processing, exosome extraction and resuspension were the same as those in Example 1.

[0094] Extraction of total RNA from rat plasma exosomes by Trizol method.

[0095] 1) Add an appropriate amount of lysis solution according to the amount of exosome precipitation, and fully mix and lyse.

[0096] 2) Transfer the above Trizol lysate to a new centrifuge tube and let it stand at room temperature for 5 minutes.

[0097] 3) In the above centrifuge tube, add chloroform accord...

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Abstract

The invention discloses a plasma exosome extraction reagent, and particularly discloses a plasma exosome extraction reagent, an enrichment method, an extraction kit and application thereof. Compared with traditional ultra-high-speed centrifugation, the technical scheme disclosed by the invention has the advantages that the pressure on the exosome in the sample is small, and a relatively complete form can be kept; according to the scheme, an amino acid polymer with positive charges, namely polylysine, poly-lysine PEG Conj [mu] gate and sulfide TCEP or DTT are added on the basis of a traditional nonionic polymer PEG, so that the time required by the extraction process is shorter, the required initial sample quantity is lower, the extraction efficiency is higher, and the extraction cost is low. The plasma exosome obtained by the scheme can be suitable for various downstream experiments, such as RNA analysis, high-throughput sequencing, cell co-culture and the like.

Description

technical field [0001] The invention discloses a plasma exosome extraction reagent, and specifically discloses a plasma exosome extraction reagent, an enrichment method, an extraction kit and applications thereof. Background technique [0002] Exosomes are secretory bodies with bilayer lipid membrane vesicle-like structures of about 30-150 nm in diameter. Most eukaryotic cells can form exosomes through secretion, and cells can transmit signaling molecules, including proteins, lipids, and nucleic acids, through exosomes. In intercellular signaling, exosomes can also specifically bind to target cells through ligand-receptor recognition. At present, there are mainly the following methods for exosome extraction: ultracentrifugation, magnetic bead immunoassay, and ultrafiltration. [0003] 1. Ultracentrifugation is the most commonly used exosome extraction method. The main operation steps are as follows: (1) low-speed centrifugation (300×g) to remove cells; (2) high-speed cent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 韩俊东李民强周丽丽
Owner 大连博格林生物科技有限公司
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