CN115200969A - Method and kit for efficiently separating and purifying plasma exosomes - Google Patents
Method and kit for efficiently separating and purifying plasma exosomes Download PDFInfo
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- CN115200969A CN115200969A CN202111140786.3A CN202111140786A CN115200969A CN 115200969 A CN115200969 A CN 115200969A CN 202111140786 A CN202111140786 A CN 202111140786A CN 115200969 A CN115200969 A CN 115200969A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation
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Abstract
The invention relates to the field of scientific research and clinical research of biological medicines, in particular to a method for efficiently separating and purifying exosomes. The invention aims to provide a reagent for separating exosomes in plasma with high quality and extracting nucleic acid molecules contained in the exosomes.
Description
Technical Field
The invention relates to the field of scientific research and clinical research of biological medicines, in particular to a method for efficiently separating and purifying exosomes.
Background
Exosomes (exosomes) are members of a vesicle family secreted by most cells, with lipid bilayer membrane structures, with diameters between 30-150nm, and differ widely in the number of exosomes secreted by different tissues or cells and the number of RNA molecules contained within the exosomes. A large amount of exosomes exist in various body fluids, and the contents of blood, hydrops in vivo, urine, milk and the like are rich. Exosomes can circulate in vivo through blood and transfer nucleic acid molecules carried in the exosomes to different tissues and cells, and therefore, the related research on the exosomes and nucleic acids contained in the exosomes has very important significance. However, the existing plasma exosome separation technology is not mature enough, and the exosome separation purity is not high enough, so that the basic and clinical research is difficult to be carried out very smoothly, and therefore, a method for separating and purifying exosomes with high efficiency and high purity is urgently needed. At present, methods for separating exosomes mainly adopt modes such as ultracentrifugation, differential centrifugation and gradient centrifugation, but the modes have high requirements on sample size, instruments and the like, so that conventional laboratories are difficult to successfully complete, and the purity, the quantity, the integrity and the like of the exosomes are influenced.
Disclosure of Invention
The invention aims to provide a reagent for separating exosomes in plasma with high quality and extracting nucleic acid molecules contained in the exosomes.
The reagent provided by the invention is a hydrophilic polymer, a salt ion solution and glycogen.
In the reagent, the hydrophilic polymer is polyethylene glycol (PEG) and the molecular weight is 6000 daltons.
In the reagent, the solvent of the salt ion solution is sterilized water, and the solute is inorganic salt sodium chloride (NaCl).
In the above reagent, the glycogen is commercially available glycogen and the concentration is 20mg/ml.
In the reagent, the hydrophilic polymer, the salt ion solution and the glycogen are mixed by 16g of polyethylene glycol, 5.84g of sodium chloride, 50mg of glycogen and 100mL of pure water.
In the reagent, the concentration of the hydrophilic polymer is 160mg/mL, and the volume is 100mL.
In the reagent, the concentration of the salt ion solution is 58.4mg/mL, and the volume is 100mL.
In the reagent, the final concentration of glycogen is 0.5mg/mL.
Of the above reagents, the mixed solution was filtered through a 0.44 μm filter to prepare a stock solution, which was designated as ExoBY.
It is another object of the present invention to provide a kit for isolating exosomes in plasma.
The kit provided by the invention, including the reagent or the application of the reagent formula, is within the scope of the invention.
In the method, the separated exosome is further detected and nucleic acid extraction and detection are carried out
The method comprises the following specific steps:
and (3) mixing the sample to be detected and the storage solution in equal volume to obtain a mixed solution, keeping the concentration of polyethylene glycol in the mixed solution at 80mg/ml, standing the mixed solution, and collecting the precipitate to obtain the exosome.
The standing condition is 4 deg.C and the time is more than 12 hr.
Relevant experiments prove that the efficiency of exosome separation can be improved by using the hydrophilic polymer polyethylene glycol (PEG) and salt ions in a matching way. The invention provides a kit for separating exosomes, which can change the microenvironment of exosomes in plasma liquid and is beneficial to sedimentation and enrichment of exosomes in the environment; the obtained exosome has high purity and a universal structure, and is suitable for the next detection of exosome and the sequencing and analysis of inclusion. The exosome separated by the invention can be extracted by a conventional nucleic acid extraction method such as TRizol, miRNA extraction kit and the like.
Drawings
Fig. 1 to 4 show the results of electron microscope detection of plasma exosomes separated by the kit of the present invention.
FIGS. 5 to 8 show the results of particle size range detection of plasma exosomes isolated by the kit of the present invention.
FIG. 9 shows the result of surface protein marker detection of plasma exosomes separated by the kit of the present invention.
FIGS. 10 to 13 show the total RNA content of the extracted contents after the plasma exosomes are separated by the kit of the present invention.
Detailed Description
The experimental methods used in the following examples are all conventional methods.
Materials, reagents, instruments and the like used in the following examples are conventional commercial products.
The hydrophilic polymer concentration units used in the following examples are mass/volume, calculated as solute (g)/solvent (mL).
The reagents, materials and instrument types used in the following examples are: PEG6000 (Sigma, 81260), nacl (Shanghai-derived leaves, S24119), glycogen (Beyotime, D0812), centrifuge (Eppendorf, 5425R).
Example 1: separating plasma exosomes and extracting content RNA.
1. Plasma sample preparation.
Using an EDTA anticoagulant blood collection tube to collect peripheral blood samples of 4 volunteers, respectively naming the samples as WR182580S, WR182582S, WR182584S, WR182586S, placing in a refrigerator at 4 ℃ for 3 hours, 4000Xg, centrifuging for 10 minutes, centrifuging cell debris, impurities and the like to the bottom of the tube, taking supernatant, and transferring to a new centrifuge tube.
2. Exosomes were isolated from plasma samples.
1. And taking 1ml of the separated plasma sample, adding 1ml of the stock solution ExoBY with the same volume, and evenly mixing by reversing to obtain the hydrophilic polymer with the final concentration of 80mg/ml and the volume ratio of 8%. Mixing, turning upside down gently, mixing, and placing into 4 deg.C refrigerator for low temperature polymerization for more than 12 hr.
2. Centrifuging the mixed solution after the low-temperature standing in a centrifuge at 4 ℃ at 10000Xg for 1 hour, removing supernatant, and precipitating at the bottom of a centrifuge tube to obtain the exosome.
3. The exosomes were resuspended in PBS and stored at-80 ℃ in a freezer for future use.
3. Exosome RNA content detection
And (3) extracting RNA from the separated and purified exosome by a TRizol method, detecting the concentration of the extracted RNA by using a Qubit, and detecting the RNA quality by using a labchip GX touch nucleic acid automatic electrophoresis apparatus according to the instruction. The RNA concentrations are shown in Table 1 and FIGS. 10 to 13:
4. and (4) identifying exosomes.
1. Exosome electron microscope identification morphological appearance
Dripping 5 μ L each of WR182580S, WR182582S, WR182584S, WR182586S exosome sample on carbon supporting film copper net, placing for 3-5min, and then sucking off redundant liquid by using filter paper; dropping 2% phosphotungstic acid on the carbon supporting film copper net, standing for 2-3min, sucking off excessive liquid with filter paper, and drying at room temperature; observing under a transmission electron microscope, and collecting and analyzing images. The electron microscope result shows that the vesicle has obvious membrane structure and the direct range is 30-200 nm. As described in fig. 1 to 4.
2. Exosome diameter range was analyzed by Electrophoresis & Brownian Motion Video Analysis
The instrument parameters adopt CA16-122-0096, the temperature is 26 ℃, the volume of WR182580S, WR182582S, WR182584S, WR182586S exosome solution is 5 mu L, the detection result shows that the particle size range of exosome is 30-200nm, and the highest peak is concentrated at about 100 nm.
3. Western blot detection of exosome surface protein
WR182580S, WR182582S, WR182584S, WR182586S was added with 20ul of lysate (100 xPMSF, 100xcocktail and phosphatase inhibitor were added in advance), mixed well and then lysed on ice for 15min, 13000g, centrifuged at 4 ℃ for 10min, and the supernatant was taken. After calculating the volume of the sample according to the protein concentration determined by the BCA method, 5 XSDS-loading buffer was added and boiled at 100 ℃ for 10min. Clamping a glass plate (1.0 mm or 1.5 mm) filled with polyacrylamide gel as required, quickly adding the prepared SDS polyacrylamide separation gel (8%, 10% or 12%) into the gap of the glass plate to 2/3 of the position of the glass plate, sealing the glass plate with isopropanol, and then performing electrophoresis detection. And after electrophoresis, carrying out steps of membrane conversion, sealing, washing, primary antibody combination, washing, secondary antibody combination, color development and the like to detect the surface protein.
Claims (9)
1. The invention provides a method for efficiently separating and purifying plasma exosomes, which is characterized by utilizing a mixed solution of a hydrophilic polymer, salt ions and glycogen.
2. The reagent of claim 1, wherein the hydrophilic polymer is polyethylene glycol (PEG) and has a molecular weight of 6000 daltons.
3. The reagent of claim 1, wherein the salt ion is NaCl at a concentration of 1M.
4. The reagent of claim 1, wherein the glycogen concentration is 20mg/mL.
5. The reagent according to claim 1, wherein the exosome is isolated at a concentration of 8% by volume and at a concentration of 160mg/mL.
6. The kit provided by the invention is characterized by comprising the reagents and characteristics of the claims 1 to 5.
7. The invention can improve the efficiency of exosome separation by using the hydrophilic polymer polyethylene glycol (PEG) and salt ions in a matching way.
8. The invention provides a kit for separating exosomes, which can change the microenvironment of the exosomes in plasma liquid and is beneficial to sedimentation and enrichment of the exosomes in the environment.
9. The invention can provide high-quality exosome in separated plasma, and the separated exosome can be subjected to nucleic acid extraction by using a conventional nucleic acid extraction method such as TRizol, miRNA extraction kit and the like.
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| CN202111140786.3A CN115200969A (en) | 2021-09-28 | 2021-09-28 | Method and kit for efficiently separating and purifying plasma exosomes |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586952A (en) * | 2024-01-19 | 2024-02-23 | 首都医科大学宣武医院 | Separation reagent and separation method for separating plasma exosomes |
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2021
- 2021-09-28 CN CN202111140786.3A patent/CN115200969A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586952A (en) * | 2024-01-19 | 2024-02-23 | 首都医科大学宣武医院 | Separation reagent and separation method for separating plasma exosomes |
| CN117586952B (en) * | 2024-01-19 | 2024-04-26 | 首都医科大学宣武医院 | Separation reagent and separation method for separating plasma exosomes |
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