CN116426606A - Rapid extraction kit and extraction method for exosomes and RNA (ribonucleic acid) in trace samples - Google Patents
Rapid extraction kit and extraction method for exosomes and RNA (ribonucleic acid) in trace samples Download PDFInfo
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Abstract
The invention discloses a kit and a method for rapidly extracting exosomes and RNA (ribonucleic acid) in a trace sample. The method is based on an immunoaffinity capturing technology to rapidly extract exosomes and exosome RNA, breaks through the prior technical bottleneck, can efficiently purify exosomes from 100 mu L trace samples, reduces exosome loss, obtains exosomes with high purity, simultaneously can further extract RNA in situ by exosomes enriched in plate holes, overcomes the technical challenges of complicated operation steps and easy degradation, and improves the yield of RNA. The invention has the advantages of low sample demand, high purity, good stability, high efficiency, low cost, no need of special instruments and equipment and the like, and provides stable and reliable technical support for the research of the related molecular level of the exosome.
Description
Technical Field
The invention relates to the technical field of biology, in particular to exosomes and extraction of exosome RNA.
Background
Exosomes (EVs) are nanoscale vesicles secreted by a variety of cells into body fluids. Its production involves the invagination of the cell membrane, the formation of intracellular multivesicular bodies (MVBs) containing luminal vesicles, which are finally released into the extracellular environment after fusion with the plasma membrane. These exosomes contain different types of proteins, lipids, RNA, DNA, etc., which mediate the transfer of information between cells, playing an important role in regulating the processes of immunity, inflammation, cell and tissue regeneration, etc.
Among them, the detection of exosome RNA has important application value in the medicine field. Included in exosomes are a variety of RNAs such as miRNAs, fragmented or intact mRNAs, ribosomal RNAs (rRNAs), and long non-coding RNA (lncRNA) molecules, among others. It was found that these RNAs might promote tumor development, invasion and increase drug resistance by promoting cell migration, invasion, colony formation, angiogenesis, immunosuppression, apoptosis inhibition, angiogenesis promotion, autophagy regulation and other mechanisms. For example, exosomes miR-23a from hypoxic lung cancer cells can increase angiogenesis and vascular permeability, thereby potentially promoting tumor cell development; exosomes mir-20b-5p and mir-3187-5p are associated with NSCLC; the absence of exosomes miR-26a-5p might contribute to endometrial cancer lymphangiogenesis and lymphatic metastasis. Therefore, exosome RNA-based liquid biopsies are expected to be a key breakthrough in future medicine.
However, the main rate limiting step in the clinical transformation of exosome RNA detection technology is the lack of efficient exosome RNA extraction methods to date. Currently common methods, such as ultracentrifugation, including differential centrifugation and density gradient centrifugation, are the most classical exosome extraction methods. However, a major problem in the inability of these methods to achieve clinical transformations is the large sample size required. Typically, more than 10mL of blood is taken for a single test to extract the amount of RNA required for conventional reverse transcription. In addition, the exosome solution obtained by extraction is easy to mix with various impurities, and the method is only applicable to lipoproteins, extracapsular protein complexes, aggregates and other substances at present, and the RNA detection result is unstable. The magnetic bead adsorption method can realize the efficient capture of the exosome subgroup through the exosome target antibody. In addition, the precipitation method is simple to operate, does not need special technology and equipment, has high yield, can be used for downstream analysis experiments, but can precipitate hydrophobic substances and other proteins of non-exosomes, and the purity of the obtained exosomes is not high. Ultrafiltration, including size-based filtration, size exclusion chromatography, and polymer precipitation, is a size-based exosome separation method, by defining a molecular weight or size exclusion limited membrane filter to separate exosomes; and microfluidic-based exosome separation techniques, are necessary and important techniques for microscale separation, detection and analysis of exosomes. However, the clinical transformation of the technology has the defects and problems of higher cost, need of matched instruments and equipment, low recovery rate, low speed and the like.
Disclosure of Invention
The invention aims to provide a rapid extraction kit and an extraction method for exosomes and RNA thereof in a trace sample, so as to solve the problems that the current exosome RNA detection sample needs to be excessively large in collection amount, is complex to operate and is easy to be degraded by RNase, and the concentration and purity of the extracted RNA are poor. Thus, stable and reliable technology and data support are provided for future research.
One of the technical schemes adopted for solving the technical problems is as follows:
an extraction kit for exosomes, the kit comprising a microplate coated with exosome capture antibodies, the microplate having a three-layer polymer coating thereon, the three-layer polymer being carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer, the three-layer polymer having exosome capture antibodies bound thereto.
Further, the three-layer polymer coating, namely carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer, is prepared by the following method:
1) Coating an epoxy-modified microplate with a partially carboxylated PAMAM dendrimer;
2) Adding 1-3 kDa methoxy-PEG-amine (mPEG), 4-6 kDa carboxyl-PEG-amine and 18-22 kDa carboxyl-PEG-amine into the product obtained in the step 1), and enabling a PEG (polyethylene glycol) mixture formed by the three PEGs to form conjugation with PAMAM and the rest epoxy groups;
3) And 2) adding the partially carboxylated PAMAM dendritic polymer into the product obtained in the step 2) again to form a carboxylated PAMAM dendritic polymer-PEG mixture-carboxylated PAMAM dendritic polymer three-layer polymer coating structure on the micro-porous plate.
Further, in the process of preparing the three-layer polymer coating, activation with EDC, NHS, etc. is required.
Further, the mass ratio of the methoxy-PEG-amine with the molecular weight of 1-3 kDa, the carboxyl-PEG-amine with the molecular weight of 4-6 kDa and the carboxyl-PEG-amine with the molecular weight of 18-22 kDa is 45-50:1:1.
Further, reacting PAMAM dendrimer with succinic anhydride in dimethyl sulfoxide to carry out amino carboxylation, so as to obtain the carboxylated PAMAM dendrimer. Specifically, after PAMAM dendrimers are purified by phosphate buffered saline and water, the mass ratio of the PAMAM dendrimers to succinic anhydride is 1: the partially carboxylated PAMAM dendrimer is obtained by reacting in DMSO at a ratio of 0.30 to 0.35 overnight (e.g. 10 to 15 h).
Further, the exosome capture antibodies bound on the tri-layer polymer of the microplate are monoclonal antibodies specific for human total exosome-associated antigens, including, for example, at least one of anti-CD 63 antibodies, anti-CD 9 antibodies, or anti-CD 81 antibodies.
Further, the exosome capture antibody coated microplate was prepared by the following method: and (3) treating the exosome capture antibody with EDTA and cysteine hydrochloric acid to obtain a modified antibody, adding the modified antibody into a microporous plate which is treated by Tween-20 and activated by SMCC and provided with a three-layer polymer coating, incubating to complete coupling of the exosome capture antibody, washing the plate, blocking with BSA, and washing to obtain the exosome capture antibody. And then used for capturing exosomes.
The second technical scheme adopted by the invention for solving the technical problems is as follows:
an exosome RNA extraction kit comprises a microplate coated with an exosome capture antibody and an RNA extraction reagent; the microplate is provided with a three-layer polymer coating, wherein the three-layer polymer is carboxylated PAMAM dendritic polymer-PEG mixture-carboxylated PAMAM dendritic polymer, and the three-layer polymer is combined with an exosome capture antibody.
Further, the three-layer polymer coating, namely carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer, is prepared by the following method:
1) Coating an epoxy-modified microplate with a partially carboxylated PAMAM dendrimer;
2) Adding 1-3 kDa methoxy-PEG-amine, 4-6 kDa carboxyl-PEG-amine and 18-22 kDa carboxyl-PEG-amine into the product obtained in the step 1), and enabling a PEG mixture formed by three PEGs to form conjugation with PAMAM and the residual epoxy groups;
3) And 2) adding the partially carboxylated PAMAM dendritic polymer into the product obtained in the step 2) again to form a carboxylated PAMAM dendritic polymer-PEG mixture-carboxylated PAMAM dendritic polymer three-layer polymer coating structure on the micro-porous plate.
Further, in the process of preparing the three-layer polymer coating, activation with EDC, NHS, etc. is required.
Further, the mass ratio of the methoxy-PEG-amine with the molecular weight of 1-3 kDa, the carboxyl-PEG-amine with the molecular weight of 4-6 kDa and the carboxyl-PEG-amine with the molecular weight of 18-22 kDa is 45-50:1.
Further, reacting PAMAM dendrimer with succinic anhydride in dimethyl sulfoxide to carry out amino carboxylation, so as to obtain the carboxylated PAMAM dendrimer. Specifically, after PAMAM dendrimers are purified by phosphate buffered saline and water, the mass ratio of the PAMAM dendrimers to succinic anhydride is 1: the partially carboxylated PAMAM dendrimer is obtained by reacting in DMSO at a ratio of 0.30 to 0.35 overnight (e.g. 10 to 15 h).
Further, the exosome capture antibodies bound on the tri-layer polymer of the microplate are monoclonal antibodies specific for human total exosome-associated antigens, including, for example, at least one of anti-CD 63 antibodies, anti-CD 9 antibodies, or anti-CD 81 antibodies.
Further, the exosome capture antibody coated microplate was prepared by the following method: and (3) treating the exosome capture antibody with EDTA and cysteine hydrochloric acid to obtain a modified antibody, adding the modified antibody into a microporous plate which is treated by Tween-20 and activated by SMCC and provided with a three-layer polymer coating, incubating to complete coupling of the exosome capture antibody, washing the plate, blocking with BSA, and washing to obtain the exosome capture antibody. And then used for capturing exosomes.
In the exosome extraction kit or exosome RNA extraction kit, a novel dendritic surface structure-loaded capture antibody is used first, so that the problem of steric hindrance of exosome capture is solved, and efficient and high-specificity exosome capture is realized. In particular, the present invention utilizes the surface of a bilayer Polyamidoamine (PAMAM) dendrimer to effectively mediate the multivalent binding effect of hyperbranched nanoparticles according to the characteristic that the sensitivity and specificity of an exosome immunoaffinity device can be significantly enhanced by the nanostructure polymer surface, while simultaneously linking a variety of antibodies to a micelle suitable for exosome nanosize through a high density of functional groups, forming an aperture plate (as in fig. 1) with sufficient surface area to accommodate the reorientation of the binding domain for efficient, specific capture of exosomes. During detection, samples such as plasma and the like can be directly added to the bottom of the coated microplate without any pretreatment. Through the immunoadsorption, exosomes in the sample are captured and fixed at the bottom of the microporous plate in one step.
When the extraction kit of the exosome RNA is used, the exosome RNA is recovered by directly cracking an ELISA plate, and the damage to unstable RNA caused by complex operations such as centrifugation, separation, recovery, cleaning and the like is skipped, so that the quality and efficiency of RNA extraction are improved. The immunoadsorption and the molecular biological diagnosis are connected in series, so that the complexity of operation is reduced, and the rapid detection of the exosome RNA from a trace plasma sample is realized (as shown in figure 2).
The third technical scheme adopted by the invention for solving the technical problems is as follows:
in the extraction method of exosomes by using the extraction kit, a sample is added into a microplate coated with exosome capture antibodies, and exosomes in the sample are captured in the microplate by immunoadsorption.
Further, the sample comprises at least one of plasma, cell supernatant, or tissue.
The fourth technical scheme adopted for solving the technical problems is as follows:
the method for extracting the exosome RNA by using the extraction kit comprises the steps of adding a sample into a micro-pore plate coated with an exosome capture antibody, and capturing the exosome in the sample in the micro-pore plate through immunoadsorption; subsequent lysis is performed in microwells and the exosome RNA recovered.
Further, the sample comprises at least one of plasma, cell supernatant, or tissue.
Further, the method for performing cleavage in a microplate and recovering the exosome RNA comprises: adding a lysate into a microplate which captures exosomes, standing to completely separate a nucleic acid-protein complex, centrifuging to obtain a supernatant, adding chloroform, transferring an obtained aqueous phase containing RNA, adding 70-80% ethanol, uniformly mixing, freezing, precipitating, and dissolving and precipitating to obtain exosome RNA.
The method is based on immune affinity capture technology to rapidly extract exosomes and exosome RNA, breaks through the bottleneck of the prior art, can efficiently purify exosomes from 100 mu L trace plasma samples, has high purity of the obtained exosomes, and can further extract RNA in situ by the exosomes enriched in plate holes. In a word, the invention has the advantages of low sample demand, high purity, good stability, high efficiency, low cost, no need of special instruments and equipment, and the like.
The microplate disclosed by the invention is preferably an ELISA plate, and has good binding capability with molecules such as protein.
The trace sample provided by the invention is that the extraction of exosomes or exosome RNAs can still be realized even if the sample volume is as low as 100 mu L.
The methods of the invention are not useful for disease diagnosis or treatment.
The equipment, reagents, processes, parameters, etc. according to the present invention are conventional equipment, reagents, processes, parameters, etc. unless otherwise specified, and are not exemplified.
All ranges recited herein are inclusive of all point values within the range.
In the present invention, the "room temperature" is a conventional ambient temperature, and may be 10 to 30 ℃.
Compared with the background technology, the technical proposal has the following advantages:
the invention provides a high-efficiency kit and a method for extracting and detecting exosomes and exosome RNA, which can be rapidly completed by using samples such as trace plasma, and the like, and the exosomes are captured through immunoadsorption, so that the exosome loss is reduced, and the purity is improved; through further in-situ tandem RNA extraction, the technical challenges of complicated operation steps and easy degradation are overcome, and the RNA yield is improved. The time cost and the sample size cost of the whole process are greatly reduced compared with the conventional centrifugal extraction. Thereby realizing the purpose of efficiently extracting the exosome RNA from the trace sample. The development of the kit and the extraction method provides stable and reliable technical support for exosome related molecular level research.
Drawings
FIG. 1 is a schematic diagram of exosome-specific capture according to the present invention.
FIG. 2 shows an RNA extraction process in an embodiment of the present invention.
FIG. 3 is a graph comparing the effect of the present invention on extracting exosomes and RNAs from per mL of plasma by conventional centrifugation. Wherein: A. number of exosomes; rna content; rna purity; qpcr detection effect.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The embodiment of the invention adopts the detailed parameters of the materials:
(1) Preparation of an ELISA plate for exosome capture:
in the first step, an elisa plate with a three layer polymer (carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer) coating was prepared. Specifically:
carboxylation of nanoparticles: the 7 th generation PAMAM dendrimer dissolved in 5% methanol was purified 5 times with Phosphate Buffered Saline (PBS) and 5 times with Double Distilled (DDI) water, respectively, on a 10000MWCO filter centrifuge. Further, 1mg of PAMAM dendrimer was reacted with 0.308mg of succinic anhydride in Dimethylsulfoxide (DMSO) overnight for aminocarboxylation and lyophilization to yield partially carboxylated G7 PAMAM dendrimer.
Preparing a three-layer polymer coating: to the plate wells of the epoxy-modified ELISA plate, 0.1mg mL of a 1.5M potassium phosphate solution (pH 11) was added -1 The bottom surface of the wells was treated overnight with partially carboxylated G7 PAMAM dendrimer. The bottom surface of the wells was activated with 15mM EDC (1-ethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 25mM NHS (N-hydroxysuccinimide), then 0.48mg mL of HBSS (Hank's balanced salt solution) was added to the wells -1 2kDa mPEG，0.01mg mL -1 5kDa carboxy-PEG-amine and 0.01mg mL -1 The 20kDa carboxy-PEG-amine was incubated for at least 4 hours. After the incubation, EDC and NHS formulated with MES (morpholinoethanesulfonic acid buffer) were added to reactivate the bottom surface of the wells. Then add a second layer of 0.1mg mL formulated with HBSS to the plate well -1 And (3) partially carboxylating the G7 PAMAM dendritic polymer, treating the surface of the hole bottom overnight, and forming a three-layer polymer coating on the ELISA plate.
In the second step, before extracting exosomes, exosome capture antibodies are coupled in the resulting elisa plate with three layers of polymer coating, for example using the conventional exosome adsorption antibodies such as anti-CD 63, CD9 and CD81 antibodies as capture antibodies, specifically:
antibody modification: with a solution containing 5mM ethylenediamine tetraacetic acid (EDTA) and 3mg mL -1 Cysteine hydrochloride PBS anti CD63, CD9 or CD81 antibody capture antibody dilution to a concentration of 0.5 u g mL -1 . The solution was treated at 37 ℃ for 30 minutes and then purified by centrifugation using a 10,000mwco filter (Amicon) immediately prior to surface conjugation. Antibody was used in 5. Mu.g mL -1 Is resuspended in PBS with EDTA to give the modified antibody.
Antibody coupling: washing the surface of the plate hole of the ELISA plate with the three-layer polymer coating obtained in the first step, treating with Tween-20, activating with SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester), and incubating with the modified antibody at 4 ℃ overnight to complete the coupling of the exosome capture antibody on the ELISA plate with the three-layer polymer coating. Prior to the exosome capture experiments, the surface was blocked with 0.5% bsa for 1 hour to prevent non-specific adsorption.
Thus, an ELISA plate for exosome capture was obtained, the bottom surface of which comprises three layers of polymers: carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer, and exosome capture antibodies were conjugated, and the use of a combination of Tween-20 and BSA in the preparation process minimized non-specific binding while providing multivalent and high flexibility in antibody orientation.
Thirdly, exosome capture is carried out.
(2) Lysate: the main components are as follows: phenol, which cleaves and releases DNA, RNA, and denatures proteins; 8-hydroxyquinoline, guanidine isothiocyanate and beta-mercaptoethanol can inhibit RNase.
(3) Other reagents and equipment: double distilled water (DDI water), RNase Free water, RNase zap, RNase Free EP tube, RNase Free cartridge head cartridge (avoiding gun interference), RNase Free centrifuge tube 50mL (no enzyme sterile for split charging), chloroform, isopropanol, 75% ethanol, soxhaust nucleic acid precipitation aid (acrylic Carrier).
(4) Washing liquid: is configured from 1 XPBST and 0.05% Tween 20.
Example 1: immune adsorption capturing method for extracting exosomes in human micro-plasma sample
(1) As described in the second step above, the surface was blocked with 0.5% bsa for 1 hour to prevent non-specific adsorption prior to the exosome capture experiment. After closing, the closing plate film is carefully removed, the liquid is discarded, and the mixture is beaten to dryness on absorbent paper.
(2) 300. Mu.L of 1 Xwashing solution was added to each well, left to stand for 1min, the solution was discarded, and the mixture was dried on absorbent paper and repeated 2 times.
(3) The plasma sample to be tested is placed on ice, centrifuged at 12,000Xg for 10min at 4℃and the supernatant is taken to remove cells or cell debris.
(4) 100. Mu.L of the centrifuged plasma sample was added to each well.
(5) The plates were sealed and incubated at 37℃for 2h.
(6) Carefully remove the sealing plate film, discard the liquid and take a pat dry on absorbent paper.
(7) 300. Mu.L of 1 Xwashing solution was added to each well, left to stand for 1min, the solution was discarded, and the mixture was dried on absorbent paper by beating, and repeated 3 times. And (5) capturing the exosomes on the ELISA plate.
Example 2: extraction detection and QPCR detection of exosome RNA in human micro plasma sample
1. Exosome extraction was performed as in example 1, with the exosome RNA to be detected being 221-5p mRNA (i.e., miRNA-221-5 p), and the capture antibody used being CD9.
2. Extraction of RNA
(1) After the exosomes were adsorbed on the bottom of the ELISA plate by the method of example 1, 200. Mu.L/well of lysate was added, followed by blowing 100 times/well, standing for 5min, shaking and mixing for 2min, blowing 100 times/well, standing for 5min, and transferring to a new EP tube.
(2) Subsequently, 200nM of external reference 5. Mu.L (10 pmol) was added to the transferred EP tube and mixed by blowing. The supernatant was centrifuged at 12000rpm for 10min and the EP tube (without enzyme) was removed.
(3) 200. Mu.L of chloroform was added to each tube, shaken for 15s, allowed to stand at room temperature for 10min, and centrifuged at 12000rpm for 15min.
(4) After centrifugation the contents of the tube were separated into three layers (not visible) and the upper aqueous phase was carefully aspirated 300. Mu.L and transferred to a fresh EP tube.
(5) Adding equal volume (300 μl) of isopropanol, mixing, slightly turning over, mixing, standing at 4deg.C for 10min, standing at-20deg.C for 1 hr, taking out, and slowly and smoothly removing the precipitate.
(6) The centrifuge is pre-cooled to 4 ℃, and the water bath is pre-heated to 55 ℃.
(7) Centrifuge at 12000rpm for 15min at 4℃during which 500. Mu.L (375. Mu.L ethanol+125. Mu.L DEPC water) of 75% ethanol was freshly prepared with DEPC water.
(8) Centrifuging, discarding the supernatant, taking trace RNA as total RNA at the bottom of the centrifuge tube at the outer side of the centrifuge tube, carefully adding 300 mu L of 75% ethanol which is prepared at present after the supernatant is completely absorbed at the bottom of the inner side tube, washing the precipitate after turning over, and standing for 1-2 min for full contact.
(9) The supernatant was carefully aspirated with a 200. Mu.L tip at 4℃and 12000rpm for 5min.
(10) Centrifuge at 10000rpm for 5min at 4℃and carefully suck off residual ethanol with a 10. Mu.L tip.
(11) Drying and concentrating: drying at room temperature for 1-2 min.
(12) RNA concentration and 260/280 data detection were performed using a nanodrop instrument.
Example 3: extraction and detection of exosome RNA in cell supernatant
100. Mu.L of the centrifuged plasma sample of example 1 was replaced with 100. Mu.L of the pretreated cell supernatant enriched sample, and the rest of the procedure was the same as in example 1.
Example 4: detection of extracted exosome concentration
1. Exosome extraction as in example 1
2. Detection of exosome concentration
(1) Pretreatment of a standard substance: exosome concentration in the standard used: 7.3X10 10 Particle count/mL, placing the standard substance in a refrigerator at 4 ℃, thawing, shaking, mixing uniformly, performing instantaneous centrifugation, and obtaining the concentration of the exosome subjected to gradient dilution according to the volume fraction of A, B, C, D, E, F groups to be added for preparing a standard curve.
(2) Adding liquid and incubating: the HRP-labeled streptavidin was diluted 1000-fold with sample dilution (which may be prepared in advance at the time of previous incubation, placed in ice) and 100 μl was added to each well and the plate was closed. Incubate at 37℃for 30min in the absence of light.
(3) Washing: after the liquid is discarded, the mixture is beaten to dryness on the absorbent paper, 300 mu L of 1 Xwashing liquid is added into each hole, the mixture is kept stand for 1min, and after the liquid is completely discarded, the mixture is beaten to dryness on the absorbent paper, and the mixture is repeated for 5 times.
(4) Color development: 50 mu L of a color reagent A and 50 mu L of a color reagent B are added into each hole, the mixture is gently vibrated and mixed uniformly, a sealing plate is not used, and the mixture is incubated at 37 ℃ for color development for 3-5 min in a dark place.
(5) Reading a plate: the reaction was stopped by adding 50. Mu.L of stop solution to each well, and the color was changed from blue to yellow, and the absorbance (OD) of each well at 450nm was read on a microplate reader.
(6) And (3) analyzing data results: in the quantitative determination, a standard curve is manufactured by using the standard substance diluted in the gradient manner in the step (1), the OD450 value of the corresponding OD450 value minus the OD450 value of the negative control is taken as an abscissa, the exosome concentration is taken as an ordinate, the standard curve is generated, a standard curve equation y=ax+b is obtained, the OD450 value of the measured sample is brought into the standard curve equation, and the exosome concentration in the detected sample, namely the y value, is calculated.
The results are shown as A in FIG. 3. Therefore, after the exosomes are extracted, the exosomes obtained by extraction can be verified by further adding the steps of enzyme-labeled secondary antibodies, chromogenic substrates and the like.
Example 5: detection of exosome miRNA expression level (for example, miRNA-221-5 p)
1. Extraction of exosome RNA as in example 2
2. Reverse transcription and QPCR
(1) The super clean bench was sprayed with RNase zap spray. To the obtained RNA (8 to 10. Mu.L), 2 to 4. Mu.L of enzyme-free water was directly added and the mixture was then made up to 12. Mu.L.
(2) Adding gDNARemover 1 μl, blowing with a pipette for more than 10 times, mixing thoroughly, standing at room temperature for 5min for reaction, and standing on ice. The 4 XmiRNART Buffer and miRNA RT Enzyme Mix were centrifuged rapidly.
(3) 4 XmiRNA RT Buffer 5. Mu.L was added and pre-denaturation of RNA was performed prior to reverse transcription (heating for 5min, rapidly to ice for 1 min).
(4) miRNA RT Enzyme Mix 2 mu L is added, the range of a liquid transferring gun is adjusted to 18 mu L, and the reverse transcription reaction system is prepared by blowing for 10 times.
(5) Reverse transcription reaction conditions: the reaction is carried out for 15min at 37 ℃,10 min at 42 ℃ and 3min at 95 ℃ to obtain the cDNA first strand containing all miRNAs.
(6) And (3) preparing a QPCR system according to the requirements of the QPCR kit system, and finishing the machine.
The result is shown as D in fig. 3. It follows that after RNA extraction cDNA can be obtained by further reverse transcription and the expression level of the RNA fragment of interest assessed by QPCR.
Experimental example
The 221-5p mRNA extraction and detection are carried out on 60 clinical plasma samples so as to compare and study the immunoadsorption capture method and the conventional centrifugal detection method according to the embodiment of the invention. The results show that:
from the experimental cost, including the calculated reagent consumables and machine time fees, the conventional ultracentrifugation method costs up to 360RMB for a single plasma sample extraction cost of 300 μl, whereas the method of the present embodiment only requires 62.43RMB (as in table 1). In comparison, the method of the embodiment of the invention only needs to be as follows: the sample is subjected to preliminary centrifugation (20 min), and is sealed (30 min), washed twice (10 min), added with sample (5 min), incubated (120 min), washed (15 min), and RNA extracted in situ (60 min) for 260min. By conventional ultracentrifugation, however, it is necessary to experience: subjecting the sample to preliminary centrifugation (20 min), recovering the supernatant (5 min), centrifuging (10 min), recovering the supernatant (10 min), centrifuging (30 min), filtering (30 min), recovering the supernatant (10 min), centrifuging (70 min), blowing and mixing (60 min), transferring to an RNA extraction tube (10 min), extracting RNA (60 min), and 445min in total. In addition, in the operation step of extracting exosomes, the operation time is up to 225min, but the method of the embodiment of the invention only needs 90min, and the manpower required by the ultracentrifugation method is greatly reduced (as shown in table 1).
From the aspect of the sample amount, the method of the embodiment of the invention can stably extract 7 multiplied by 10 by only 100 mu L of each blood plasma sample 11 The exosomes (as in fig. 3 a). From these exosomes, 1000-1500 ng of RNA was stably extracted, the concentration was stabilized in the interval of 40+ -5 ng/. Mu.L (as in FIG. 3B), and the OD value was also stabilized between 1.4 and 2.0 (as in FIG. 3C). The purity and the extraction amount of RNA are fully satisfied with the requirements of subsequent RNA reverse transcription, real time-qPCR and other detection (usually, the detection kit requires RNA with the minimum concentration of 8 ng/. Mu.L and OD value of 1.4-2.0). Whereas the same amount of exosomes was obtained by centrifugation, 2.6mL of plasma was required (as in fig. 3 a). However, at least 1500ng of RNA is required to be obtained300. Mu.L of plasma sample, and the OD value was not always 1.4-2.0 (as in FIG. 3B). Therefore, compared with the traditional ultracentrifugation method in which the extraction of exosomes is performed after the extraction of RNA, the extraction concentration, stability and purity of exosome RNA by the immunoadsorption capture method of the embodiment of the invention are all significantly improved (as shown in fig. 3). Compared with QPCR detection of the extracted exosome RNA, in the detection of 221-5p mRNA, the expression level of the immunoadsorption capture method of the embodiment of the invention is far higher than that of an ultracentrifugation method (as shown in figure 3D), which indicates that the exosome RNA obtained by the capture method has better experimental effect.
TABLE 1 comparison of Experimental costs and operating time costs for ultracentrifugation and immunoadsorption Capture
The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.
Claims (10)
1. An exosome extraction kit which characterized in that: the kit comprises a microplate coated with an exosome capture antibody, wherein the microplate is provided with a three-layer polymer coating, the three-layer polymer is carboxylated PAMAM dendritic polymer-PEG mixture-carboxylated PAMAM dendritic polymer, and the exosome capture antibody is combined on the three-layer polymer.
2. An exosome RNA extraction kit, characterized in that: the kit comprises a microplate coated with exosome capture antibodies and also comprises an RNA extraction reagent; the microplate has a three-layer polymer coating thereon, the three-layer polymer is carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer, and the exosome capture antibody is bound to the three-layer polymer.
3. The extraction kit according to claim 1 or 2, characterized in that: the carboxylated PAMAM dendrimer-PEG mixture-carboxylated PAMAM dendrimer three-layer polymer coating is prepared by the following method:
1) Coating an epoxy-modified microplate with carboxylated PAMAM dendrimer;
2) Adding 1-3 kDa methoxy-PEG-amine, 4-6 kDa carboxyl-PEG-amine and 18-22 kDa carboxyl-PEG-amine into the product obtained in the step 1), and enabling a PEG mixture formed by three PEGs to form conjugation with PAMAM and the residual epoxy groups;
3) And 2) adding carboxylated PAMAM dendritic polymer into the product obtained in the step 2) again to form a three-layer polymer coating structure of carboxylated PAMAM dendritic polymer-PEG mixture-carboxylated PAMAM dendritic polymer on the micro-porous plate.
4. The extraction kit of claim 3, wherein: the mass ratio of the 1-3 kDa methoxy-PEG-amine to the 4-6 kDa carboxyl-PEG-amine to the 18-22 kDa carboxyl-PEG-amine is 45-50:1:1.
5. The extraction kit of claim 3, wherein: and (3) reacting the PAMAM dendrimer with succinic anhydride in dimethyl sulfoxide to carry out amino carboxylation, so as to obtain the carboxylated PAMAM dendrimer.
6. The extraction kit according to claim 1 or 2, characterized in that: the exosome capture antibodies bound to the tri-layer polymer include at least one of an anti-CD 63 antibody, an anti-CD 9 antibody, or an anti-CD 81 antibody.
7. The extraction kit according to claim 1 or 2, characterized in that: the microplate coated with the exosome capture antibody is prepared by the following method: and (3) treating the exosome capture antibody with EDTA and cysteine hydrochloric acid to obtain a modified antibody, adding the modified antibody into a micro-porous plate which is treated by Tween-20 and activated by SMCC and provided with a three-layer polymer coating, incubating to complete coupling of the exosome capture antibody, blocking with BSA, and then capturing the exosome.
8. A method for extracting exosomes using the extraction kit of claim 1, characterized in that: the sample is added to a microplate coated with an exosome capture antibody, and exosomes in the sample are captured in the microplate by immunoadsorption.
9. A method for extracting exosome RNA using the extraction kit of claim 2, characterized in that: adding a sample into a microplate coated with an exosome capture antibody, wherein exosomes in the sample are captured in the microplate through immunoadsorption; adding a lysate into a microplate which captures exosomes for cracking, standing to completely separate a nucleic acid-protein complex, centrifuging to obtain a supernatant, adding chloroform, transferring an obtained aqueous phase containing RNA, adding 70-80% ethanol, uniformly mixing, freezing, precipitating, and dissolving and precipitating to obtain exosome RNA.
10. Extraction method according to claim 9 or 10, characterized in that: the sample includes at least one of plasma, cell supernatant, or tissue.
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| CN117210454B (en) * | 2023-11-09 | 2024-01-30 | 北京纳捷诊断试剂有限公司 | Lysate for extracting nucleic acid from whole blood sample and application thereof |
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