CN114574437A - Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof - Google Patents
Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof Download PDFInfo
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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 the traditional ultra-high speed centrifugation, the technical scheme disclosed by the invention has the advantages that the pressure on exosomes in a sample is smaller, and the complete form can be kept; according to the scheme, polylysine, namely an amino acid polymer with positive charges, 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 amount of a sample 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
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.
Background
Exosomes are secretory corpuscles with a double-layer lipid membrane vesicular structure of about 30-150nm in diameter. Most eukaryotic cells form exosomes through secretion, and the cells can transmit signaling molecules including proteins, lipids, and nucleic acids through the exosomes. Exosomes also specifically bind target cells through ligand-receptor recognition in intercellular signaling. At present, the methods for extracting exosomes mainly comprise the following steps: ultracentrifugation, magnetic bead immunization, and ultrafiltration.
1. Ultracentrifugation is the most commonly used method for exosome extraction. The main operation steps are as follows: (1) cells were removed by low speed centrifugation (300 Xg); (2) high speed centrifugation (16,500Xg) to remove cell debris; (3) removing large volume secretion corpuscles through a 0.22 mu m filter membrane; (4) the exosomes were pelleted by ultracentrifugation (120,000 Xg).
Disadvantages of the ultracentrifugation method: the time is consumed, and the whole extraction process at least needs 8 to 24 hours; the extraction efficiency is low, and the extraction efficiency of the exosome by ultracentrifugation is only 10 percent as reported in documents; the equipment requirement is high, and the conventional laboratory does not have the condition of ultracentrifugation.
2. Magnetic bead precipitation method: according to the method, the magnetic beads coupled with the exosome specific antibodies are incubated with the sample, and exosomes expressing corresponding antigens are captured and precipitated by the magnetic beads. The surface of the exosome is provided with a specific marker (such as CD63 and CD9 protein), and the exosome can be adsorbed and separated by using magnetic beads coated with anti-marker antibodies to be combined with exosome vesicles after incubation.
Disadvantages of the magnetic bead precipitation method: the cost is high, the magnetic bead coupled antibody is expensive, generally only can be used for a trace sample, and is not suitable for the exosome extraction of a large-volume sample; the extraction efficiency is low, and only exosomes which specifically express corresponding antigens can be extracted due to the specificity of the magnetic bead labeled antibody, so that the extraction effect of the method is limited;
3. and (3) ultrafiltration: ultrafiltration membranes can also be used to separate exosomes. Exosomes are separated from proteins and other macromolecules according to their size. Disadvantages of the ultrafiltration process: this method usually requires the combination of ultracentrifugation and may lose exosomes due to adhesion of the filtration membrane and the pressure and shear force at the time of filtration may deform the exosomes to be damaged.
Disclosure of Invention
In view of the above situation, the invention discloses a plasma exosome extraction reagent, an enrichment method, an extraction kit and application thereof.
The technical scheme of the invention is as follows:
a plasma exosome extraction reagent comprising a non-ionic polymer, a sulfide, with or without added salt.
Further, the plasma exosome extraction reagent is characterized in that the nonionic polymer is one or more selected from PEG, polylysine PEG, DSS, PVP and dextran sulfate; the sulfide is selected from one or more of TCEP, DTT and mercaptoethanol; the salt is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, manganese chloride, sodium monohydrogen phosphate and sodium dihydrogen phosphate.
Further, in the plasma exosome extraction reagent, the molecular weight of the PEG is 5000-; the molecular weight of PVP is 10000-; the dextran sulfate has a molecular weight of 3000-50000 dalton, specifically 5000 dalton, 10000 dalton, 25000 dalton and 40000 dalton; the molecular weight of the polylysine is 3-30 million daltons, and specifically can be 3-7 million daltons, 7-15 million daltons, 15-30 million daltons; the polylysine PEG is polylysine PEG Conj mu gate, the molecular weight is 2000 or is polylysine PEG Conj mu gate, and the molecular weight is 5000.
Further, the final mass concentration of the nonionic polymer in the plasma exosome extraction reagent is 10-50%.
Further, the reagent for extracting plasma exosomes, wherein the final concentration of the sulfide in the reagent is in a range of 0.1-10 mg/ml.
Further, the above-mentioned one plasma exosome-extracting reagent, wherein the final concentration of the salt in the reagent is in the range of 5mM to 500 mM.
Further, the method for enriching the exosomes by using the plasma exosome extraction reagent comprises the following steps:
1) obtaining a plasma sample according to a conventional method, and adding physiological saline or phosphate buffer solution to treat the sample; adding the mixture with the volume of 0.5-2 times of the sample volume, and fully and uniformly mixing;
2) adding a proteinase K solution into the biological sample obtained in the step 1), wherein the addition volume of the proteinase K solution is 0.01-0.1 time of the final volume in the step 1), and treating at 30-40 ℃ after fully and uniformly mixing; then adding a plasma exosome extraction reagent, wherein the volume of the plasma exosome extraction reagent is 0.1-2 times of the total volume.
3) Uniformly mixing the solution obtained in the step 2), and standing for 1-60 minutes at the temperature of 2-8 ℃.
4) Centrifuging the solution obtained in the step 3) at low speed for 5000-10000g of sample, wherein the centrifugation time is 5-30 minutes, and the obtained precipitate is an exosome.
Preferably, the conventional method is:
1) pretreating a blood sample, collecting blood, performing anticoagulation treatment, and standing for 1h at room temperature or standing overnight at 2-8 ℃;
centrifuging at 4 deg.C and 3000Xg for 5-10 min to obtain supernatant as blood plasma, and carefully transferring the supernatant to a new centrifuge tube;
centrifuging at 4 ℃ at 10000Xg for 20 minutes to remove residual fragments; the supernatant was transferred to a new centrifuge tube, taking care not to aspirate the sediment at the bottom;
adding physiological saline or phosphate buffer solution to process the sample; adding the mixture with the volume of 0.5-2 times of the sample volume, and fully and uniformly mixing.
A plasma exosome extraction kit comprises the plasma exosome extraction reagent.
Further, the plasma exosome extraction kit further comprises proteinase K.
Further, the plasma exosome extraction reagent is applied to extraction of mouse or rat plasma exosomes.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional ultra-high speed centrifugation, the technical scheme disclosed by the invention has the advantages that the pressure on exosomes in a sample is smaller, and the complete form can be kept; according to the scheme, polylysine, namely amino acid polymer with positive charges, poly-lysine PEG Conj mu gate and sulfide TCEP or DTT are added on the basis of the traditional nonionic polymer PEG, so that the time required by the extraction process is shorter, the required sample initial amount 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.
Drawings
FIG. 1 shows the results of western blot verification in example 1;
FIG. 2 is a graph showing the particle size distribution in example 1;
FIG. 3 is a picture of NTA film in example 1;
FIG. 4 is a graph showing the effect of exosome extraction in example 2;
FIG. 5 shows the total RNA extraction amount of plasma exosomes in example 2.
Detailed Description
The test methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Polymers of varying degrees of polymerization are available from TCI company. The CD9 antibody was purchased from Abcam. The zemer flyer product is total exosome isolation kit (from plasma) (4484450), the operation of which is performed entirely according to the instructions provided with the product. The formulations of the plasma exosome extracting agents in examples 1 and 2 are shown in table 1 below.
Table 1 formula of plasma exosome extracting agent in examples 1 and 2
Example 1
Mouse plasma exosome extraction and related detection
1. Preparation of plasma
1) Adding a certain proportion of anticoagulant into a blood collection tube in advance, slowly reversing the blood collection tube and uniformly mixing after blood collection is finished, and standing the uniformly mixed whole blood for 1h at room temperature or overnight at 2-8 ℃;
2) centrifuging at 4 deg.C and 3000Xg for 5-10 min to obtain supernatant transparent yellow liquid as plasma, transferring supernatant into new centrifuge tube, and sucking out the red blood cell component.
3) The collected blood plasma can be directly used for subsequent exosome extraction experiments or stored in a refrigerator at minus 80 ℃ after being subpackaged.
2. Preparing the sample
1) Plasma (fresh sample or frozen sample that has been thawed in a water bath at 25 ℃) is placed on ice;
2) centrifuging at 2000Xg for 20 min at 4 deg.C to remove residual cells and debris;
3) transfer the supernatant to a new centrifuge tube (take care not to suck to the bottom pellet);
4) centrifugation was carried out at 10000Xg for 20 minutes at 4 ℃ to remove residual debris.
5) The supernatant was transferred to a new centrifuge tube, and the bottom pellet was removed by pipetting and placed on ice until use.
3. Extraction of exosomes (treatment with proteinase K)
Most proteins in plasma can be removed by proteinase K, but to some extent may also cause degradation of exosome surface proteins, if this has an effect on the experiment, one may choose not to perform proteinase K treatment.
1) According to the experiment requirement, sucking a proper amount of centrifuged plasma to a new centrifuge tube, and adding 1xPBS with the volume of 0.5 time;
2) fully mixing by vortex;
3) add 0.05 volumes of proteinase K. For example, an initial plasma volume of 100ul would require 5ul of proteinase K;
4) vortex and mix well and incubate for 10 min at 37 ℃.
5) Adding 0.2 volume (total volume ═ plasma + PBS) of extraction reagent;
6) mixing the mixture upside down or blowing and beating the mixture by a pipettor until the mixture becomes a uniform solution;
note: after the extraction reagent is added, the solution is cloudy.
7) Incubating for 30 minutes at 2-8 ℃;
8) after the incubation is finished, centrifuging at 4 ℃ for 5 minutes at 10000 Xg;
note: mouse plasma, incubated followed by centrifugation at 10000Xg for 30 min.
9) Carefully remove the supernatant with a pipette and suck it clean as much as possible. The sediment at the bottom of the tube is the extracted exosome;
10) (optional step) 10000xg centrifugation for 30s to remove residual extraction reagent;
11) the supernatant was carefully aspirated and discarded, then manipulated according to the "resuspend exosome" procedure.
4. Extraction of exosomes (treatment without proteinase K)
1) According to the experiment requirement, sucking a proper amount of centrifuged plasma to a new centrifuge tube, and adding 1xPBS with the volume of 0.5 time;
2) fully mixing by vortex;
3) adding 0.2 volume (total volume ═ plasma + PBS) of extraction reagent;
4) mixing the mixture upside down or blowing and beating the mixture by a pipettor until the mixture becomes a uniform solution;
note: after the extraction reagent is added, the solution is cloudy.
5) Incubating for 10 minutes at room temperature;
6) after incubation, centrifugation was carried out at 10000Xg for 5 minutes at 4 ℃.
7) Carefully remove the supernatant with a pipette and suck it clean as much as possible. The sediment at the bottom of the tube is the extracted exosome.
8) (optional step) 10000Xg are centrifuged for 30s and the remaining extraction reagent is settled.
9) The supernatant was carefully aspirated and discarded, and then manipulated according to the "resuspend exosomes" procedure.
5. Resuspension of exosomes
1) 1xPBS or other similar buffer solution can be used, and the precipitate is fully blown and uniformly mixed by a liquid shifter;
2) storing the separated exosome at 2-8 deg.C for 1 week or less at-20 deg.C for a long time.
6. Extract exosome detection result
1) And extracting the detection of the marker protein CD9 on the surface of the plasma exosome.
Exosomes extracted from mouse plasma. 100ul plasma finally obtained exosome precipitate, RIPA lysate is added, vortex cracking precipitate is fully blown, and then 6 x protein loading buffer solution is added. 10 μ l of each well was run on a 10% SDS-PAGE. 5% skim milk was blocked for 30 min, and the primary antibody (CD9 antibody, abcam) was incubated overnight at 4 ℃ and the secondary antibody (Goat Anti-Mouse IgG, HRP Conjugate) was incubated for 1h at room temperature with ECL luminescence and color development, the results are shown in FIG. 1. As can be seen from figure 1, the marker protein detected by the exosome prepared by the exosome extraction and purification method has good specificity, has no interference of non-specific impurity protein in detection, and achieves the same effect with the semer fly. Proteinase K is required to be selected for use in an exosome extraction experiment according to the requirements of a subsequent experiment.
2) Particle size detection for plasma exosome extraction
Exosomes extracted from mouse plasma. And adding 50-100ul PBS buffer solution into the exosome precipitate finally obtained from 100ul plasma, fully resuspending the exosome, and carrying out NTA detection. The NTA result shows that the total particle size of the exosome is distributed near 100nm, and the plasma exosome extracted by the method has no difference with the extraction effect of the semelafin basically. Wherein the particle size distribution of the exosomes extracted in the present invention (central in the upper panel of figure 2) is slightly better than that of semelafin (left in the upper panel of figure 2) when protease K is not added. Fig. 3 is a NTA cine picture of a sample of plasma exosomes (mice) extracted. The particle size distribution of the total plasma exosomes extracted in the invention is more fully shown to be no different from the size distribution of the total plasma exosomes extracted in the invention. Instrument model ZetaView PMX 110(Particle Metrix, Meerbusch, Germany).
Example 2
Extraction of rat plasma exosomes and related assays
1. The sample treatment, exosome extraction and resuspension procedures were the same as in example 1.
The results in FIG. 4 show that the amount of exosome-precipitated extracted by the present invention is indistinguishable from the inlet control for the same sample size.
2. Plasma exosomes (rat) RNA extraction total comparison.
The sample treatment, exosome extraction and resuspension procedures were the same as in example 1.
Extracting total RNA of rat plasma exosomes by a Trizol method.
1) Adding a proper amount of lysis solution according to the precipitation amount of the exosome, fully blowing, uniformly mixing and lysing.
2) The Trizol lysate was transferred to a new centrifuge tube and left at room temperature for 5 minutes.
3) Adding chloroform into the centrifuge tube according to the amount of 0.2ml of chloroform added into 1ml of Trizol lysate, fully shaking and uniformly mixing for 15 seconds, standing for 2-3 minutes at room temperature, 12000g, and centrifuging for 15 minutes at 2-8 ℃.
4) The upper aqueous phase was removed from the new centrifugation audience, and isopropanol was added in an amount of 0.5ml per 1ml of Trizol lysate, left at room temperature for 10 minutes, 12000g, and centrifuged at 2-8 ℃ for 10 minutes.
5) Discarding the supernatant, adding 1ml of 75% ethanol into 1ml of Trizol lysate for washing, uniformly mixing by vortex, centrifuging at the temperature of 2-8 ℃ for 5 minutes, and discarding the supernatant.
6) The precipitated RNA was allowed to dry naturally and the RNA precipitate was dissolved with nuclease-free water.
7) The concentration of extracted RNA was measured using NanoDrop and the total amount was calculated.
The results are shown in fig. 5, 400ul rat plasma is treated by proteinase K, and then RNA is continuously extracted from the obtained exosomes and the total amount is determined by using the semelafin and the plasma exosome extraction kit of the present invention. The result shows that the total RNA extraction amount of the plasma exosomes extracted in the invention is higher than that of the semer fly, thereby being more beneficial to downstream experiments.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A plasma exosome extraction reagent comprising a non-ionic polymer, a sulfide, with or without the addition of a salt.
2. The plasma exosome extraction reagent according to claim 1, wherein the non-ionic polymer is selected from one or more of PEG, polylysine PEG, DSS, PVP, dextran sulphate; the sulfide is selected from one or more of TCEP, DTT and mercaptoethanol; the salt is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, manganese chloride, sodium monohydrogen phosphate and sodium dihydrogen phosphate.
3. The reagent for extracting plasma exosomes according to claim 2, wherein the molecular weight of PEG is 5000-; the molecular weight of the PVP is 10000-; the molecular weight of the dextran sulfate is 3000-50000 dalton; the molecular weight of the polylysine is 3-30 ten thousand daltons; the polylysine PEG is polylysine PEG Conj mu gate, and the molecular weight is 2000-5000 Dalton.
4. The plasma exosome extraction reagent according to claim 2, wherein the final mass concentration of the non-ionic polymer in the reagent is 10-50%.
5. A plasma exosome extracting reagent according to claim 2, wherein the final concentration of said sulfide in the reagent is in the range of 0.1-10 mg/ml.
6. A plasma exosome extraction reagent according to claim 2, wherein the final concentration of said salt in the reagent is in the range 5mM-500 mM.
7. A method of enriching exosomes using the plasma exosome extracting reagent of any one of claims 1-6, comprising the steps of:
1) obtaining a plasma sample according to a conventional method, and adding physiological saline or phosphate buffer solution to treat the sample; adding the mixture with the volume of 0.5-2 times of the sample volume, and fully and uniformly mixing;
2) adding a proteinase K solution into the biological sample obtained in the step 1), wherein the addition volume of the proteinase K solution is 0.01-0.1 time of the final volume in the step 1), and treating at 30-40 ℃ after fully and uniformly mixing; then adding a plasma exosome extraction reagent, wherein the volume of the plasma exosome extraction reagent is 0.1-2 times of the total volume;
3) uniformly mixing the solution obtained in the step 2), and standing for 1-60 minutes at the temperature of 2-8 ℃;
4) centrifuging the solution obtained in the step 3) at low speed for 5000-10000g of sample, wherein the centrifugation time is 5-30 minutes, and the obtained precipitate is an exosome.
8. A plasma exosome extraction kit comprising the plasma exosome extraction reagent according to any one of claims 1 to 6.
9. The plasma exosome extraction kit according to claim 8, characterized in that it further comprises proteinase K.
10. Use of a plasma exosome extracting reagent according to any one of claims 1-6 in extracting mouse or rat plasma exosomes.
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Cited By (1)
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| CN115354024A (en) * | 2022-07-29 | 2022-11-18 | 杭州昱鼎生物科技有限公司 | Blood sample exosome extraction kit based on combination of protease treatment, SEC (SEC-activated charcoal) column and membrane filtration technology and application |
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