CN110724734A - Artificial liposome containing miRNA micic and preparation method and application thereof - Google Patents
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Abstract
The invention provides an artificial liposome containing miRNA micic, and a preparation method and application thereof. The artificial liposome is formed by mixing a liposome with a lipid bilayer structure and miRNA mimic; wherein, the miRNA imic is artificially synthesized non-human miRNA and derivatives thereof. The invention utilizes the artificial liposome coated with exogenous miRNA mimic to perform the representation of the miRNA extraction efficiency of the extracellular vesicle sample and the quantitative analysis of the extracellular vesicle miRNA in the body fluid sample, thereby providing powerful technical support for applying the vesicle miRNA to the stable quantitative detection of clinical requirements.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an artificial liposome containing miRNA mimic and a preparation method and application thereof.
Background
Extracellular Vesicles (EVs) refer to vesicular bodies with a double-layer membrane structure, which are shed from cell membranes or secreted by cells, and have diameters of 30-1000nm, and mainly consist of MicroVesicles (MVs) and exosomes (exosomes), and the MicroVesicles are small Vesicles shed from cell membranes after cells are activated or damaged. Extracellular vesicles are of great interest in disease diagnosis, particularly exosomes, due to their unique biological characteristics.
The exosome is a membrane vesicle with the particle size of 30-150nm secreted into the extracellular environment after an intracellular multivesicular body and a cell membrane are fused, is an important medium for intercellular information transfer, and plays an important role in antigen presentation, apoptosis, inflammatory reaction, tumorigenesis development and metastasis processes. It is widely distributed in body fluid, including blood, saliva, urine, milk, hydrothorax and ascites, etc.; the miRNA inclusion compound can be used as a noninvasive diagnosis marker for various diseases such as tumors and the like.
The expression level of the vesicle specific miRNA in the detection sample is high and low, and the detection sample is mainly influenced by two processes of exosome separation and exosome RNA extraction. For RNA quantitative analysis, common methods for miRNA quantitative analysis mainly include an internal reference method and an external reference method. The internal reference method carries out normalization analysis through the expression quantity of the target gene and the expression quantity of the housekeeping gene with constant expression quantity, eliminates fluctuation brought by the experimental operation process, and further judges the expression abundance of the target gene. The external reference method is characterized in that artificial synthetic miRNA is artificially added in the operation process, RNA is extracted, the amount of target miRNA and external reference miRNA is simultaneously detected, the expression amount of target gene and the expression amount of external reference gene are used for normalization analysis, and interference factors introduced in the experiment process are eliminated. However, for vesicle miRNA analysis, because a recognized housekeeping gene with constant expression level is lacked as an internal reference at the present stage, the application of an internal reference method is greatly limited, and an external reference method has obvious advantages in quantitative analysis of vesicle miRNA. However, in the current vesicle miRNA analysis based on the external reference method, after vesicle separation, free artificially synthesized miRNA imici is added before RNA extraction, and finally normalization analysis is carried out. The external reference method of the free miRNA mimic can only eliminate interference factors in the RNA extraction process, but cannot eliminate interference factors in the vesicle separation stage, and is difficult to be used for the reference of marker normalization among different samples, so that the external reference method cannot be effectively used for abundance analysis of the vesicle miRNA in clinical samples.
Disclosure of Invention
The invention aims to provide an artificial liposome containing miRNA micic, and a preparation method and application thereof.
In order to achieve the purpose of the invention, in a first aspect, the invention provides an artificial liposome containing miRNA micic, which is formed by mixing a liposome with a lipid bilayer structure and miRNA micic; the miRNA micic is artificially synthesized non-human miRNA and derivatives thereof.
The raw material for preparing the liposome with the lipid bilayer structure is at least one selected from phosphatidylcholine, phosphatidylethanolamine, cholesterol, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin.
Preferably, the molar ratio of phosphatidylcholine to phosphatidylethanolamine to cholesterol in the liposome having a lipid bilayer structure is 5-8:1-3: 1-3.
In the invention, the derivative of the non-human miRNA is at least one of the following a) to c):
a) the derivative is obtained by replacing part of the base of miRNA with artificial nucleotide base;
b) transforming the framework of miRNA into a phosphorothioate framework to obtain a derivative;
c) and modifying miRNA by polyethylene glycol to obtain the derivative.
The miRNA micid can be a single miRNA or a mixture of miRNA micids.
Preferably, the miRNA mimic is a mixture of 5'-GGGUACCAUACCGGUUGUCUUA-3', 5'-UGCUACUCCGAUCUUUAGCCUC-3', and 5'-GUCCCACUCCGUAGAUCUGUUC-3' (SEQ ID NOS: 1-3).
The particle size of the artificial liposome containing miRNA micic is 30-1000nm (preferably 30-150nm), and the average density is 1.06-1.23 g/mL.
In a second aspect, the invention provides a preparation method of an artificial liposome containing miRNA micic, comprising the following steps:
1) liposomes were prepared by a constant pressure controlled extrusion method: sequentially adding 50-150 mu L of chloroform and 20-50 mu L of methanol into a glass bottle, and then sequentially adding 150-250 mu L of 10mg/mL phosphatidylcholine, 30-50 mu L of 10mg/mL phosphatidylethanolamine and 10-30 mu L of 11mg/mL cholesterol into the glass bottle; volatilizing the organic solvents chloroform and methanol with nitrogen until the formation of thin film lipids at the bottom of the vial is observed; placing the glass bottle in a vacuum drier to remove residual organic solvent; then adding 1mL of PBS buffer solution containing 0.05-5pmol of miRNA imic and 10-30 μ L of RNase inhibitor at 11mg/mL into a glass bottle; obtaining liposome with similar vesicle particle size through a filter or a filter membrane with the pore diameter of 50 nm-800 nm;
2) removal of free miRNA mimic: transferring the liposome obtained in the step 1) to a gel exclusion chromatographic column for column chromatography, and collecting fractions with the particle size of 30-1000nm (preferably 30-150 nm);
3) concentration of artificial liposomes: the fraction collected by column chromatography is concentrated by ultracentrifugation or ultrafiltration.
The electron microscope image and the identification result of the artificial liposome containing miRNA micic provided by the invention are shown in figure 1. The particle size, the density and the electron microscope image of the micro-nano-particles can be consistent with those of the vesicle, the behavior characteristics of the vesicle can be simulated in an extraction mode based on the particle size, the density and the surface charge, and the artificially synthesized miRNA micic can be detected by a Realtime PCR instrument.
In a third aspect, the invention provides any one of the following applications of the artificial liposome or the artificial liposome prepared by the method:
(1) used for evaluating the extraction efficiency of the extracellular vesicle miRNA;
(2) the method is used for quantitative analysis of the miRNA of the extracellular vesicles.
In a fourth aspect, the present invention provides a method for evaluating extraction efficiency of extracellular vesicle miRNA, the method comprising: mixing 50-200 mu L of extracellular vesicle extraction sample with 4-20 mu L of the artificial liposome with known concentration or the artificial liposome prepared by the method (the liposome with specific distribution is adopted according to the type of the vesicle to be evaluated, for example, when the exosome separation efficiency is evaluated, the fluorescent liposome with the particle size distribution of 30-150nm is adopted), carrying out vesicle separation by a specific method, then extracting RNA in the vesicle, and detecting the expression quantity of miRNA mimic by a PCR method, thus obtaining the miRNA extraction efficiency of the extracellular vesicle extraction sample.
In a fifth aspect, the present invention provides a method for quantitative analysis of extracellular vesicle miRNA, the method comprising: adding 4-20 mul of artificial liposome into each 1mL of plasma sample, or adding 4-40 mul of artificial liposome into each 20mL of urine sample, or adding 4-40 mul of artificial liposome into each 2mL of pleural and abdominal water sample, or adding 2-40 mul of artificial liposome into each 1mL of saliva sample, or adding 2-40 mul of artificial liposome into each 2mL of milk sample, mixing the sample to be detected with the artificial liposome with known concentration or the artificial liposome prepared by the method, separating the vesicles by using a specific method, extracting RNA in the vesicles, respectively detecting the expression amounts of miRNA mimic and endogenous target miRNA by using a PCR method, and comparing the expression amount of the target miRNA with the expression amount of miRNA mimic to obtain the relative expression amount of the vesicle miRNA in the sample to be detected.
Preferably, the specific method is ultracentrifugation, gradient density centrifugation, membrane affinity (qiagen exoeasy), polymer precipitation (SBI Exoquick), chromatography, or the like.
Preferably, the method for extracting vesicle RNA is isopropanol precipitation, ethanol precipitation, adsorption column method, or the like.
The PCR method used in the present invention may be real-time fluorescent quantitative PCR (qPCR) or digital PCR.
The method is particularly suitable for analyzing the expression abundance of the exosome miRNA.
In a sixth aspect, the invention provides an application of the artificial liposome or the artificial liposome prepared by the method in the extraction process of the quality control vesicle miRNA.
The invention prepares liposome by using phosphatidylcholine, phosphatidylethanolamine, cholesterol, chloroform and artificially synthesized miRNA micic by a constant pressure control extrusion method; and (3) after removing the free miRNA micic in the system, determining the particle size, the fluorescence intensity and the abundance of the miRNA micic of the liposome. Mixing the liposome with artificially synthesized miRNA mici with serum, plasma and urine, and performing vesicle separation by using different separation methods including ultracentrifugation, membrane affinity method, polymer precipitation, chromatography and the like; RNA extraction and Realtime PCR quantitative analysis were performed on the vesicles obtained by separation. The relative expression abundance of the target miRNA in the original sample vesicle can be calculated through the quantitative results of the target gene and the miRNA mimic (figure 3).
The invention utilizes the liposome containing artificially synthesized miRNA mimic to quantitatively analyze the expression abundance of vesicle miRNA in a sample, and provides powerful technical support for applying the vesicle miRNA to a stable detection system required by clinical detection. Compared with the prior art, the invention has at least the following advantages:
the invention can simultaneously eliminate the difference factors in the vesicle separation and RNA extraction processes in the vesicle miRNA quantitative analysis, and compared with the traditional external reference method of adding free miRNA mimic after the vesicle extraction, the method is more reliable and accurate in the vesicle miRNA quantitative analysis.
The invention can be used for separating and detecting the vesicle miRNA of each clinical sample, and does not influence the determination of DNA, RNA and protein.
Drawings
Fig. 1 shows the electron microscope identification result (a) and the PCR quantification result (b) of mirnammimc of the artificial liposome containing miRNA mimic in example 2 of the present invention.
Fig. 2 is a result of stability analysis of the artificial liposome containing miRNA mimic in example 3 of the present invention.
Fig. 3 is a general flow chart of the liposome-based quantitative analysis method for vesicular miRNA according to the present invention.
Fig. 4 shows the result of the extraction process for controlling the extracellular vesicle miRNA in example 4 of the present invention.
FIG. 5 shows the result of plasma vesicle miRNA quantitative analysis based on exclusion method in example 5 of the present invention.
Detailed Description
The invention provides an artificial liposome carrying miRNA micic, which is used for quantitative analysis of extracellular vesicle miRNA in a sample. The liposome with miRNA mimic is mixed with the extracellular vesicles in a certain proportion, RNA extraction is carried out on the extracellular vesicles, and the efficiency of the miRNA extraction process of each extracellular vesicle sample can be controlled qualitatively through the quantitative result of exogenous miRNA. In addition, the liposome with miRNA mici is mixed with serum, plasma and urine samples according to a certain proportion, and then different separation methods are used for vesicle separation, wherein the separation methods comprise ultracentrifugation, membrane affinity method, polymer precipitation, exclusion chromatography and other methods; RNA extraction is carried out on the vesicles obtained by separation. By simultaneously carrying out PCR detection on the target miRNA and the exogenous miRNAminic and carrying out normalization processing on the quantitative result of the target miRNA by using the quantitative result of the exogenous miRNA mimic, the relative abundance of the target gene in the sample can be calculated.
The artificial liposome carrying miRNA micic can be prepared by the following method:
1. liposomes were prepared by a constant pressure controlled extrusion method: adding 50-150 mu L of chloroform into a 20mL glass bottle by using a 250 mu L sealed glass syringe; then adding 20-50 mu L of methanol into the same glass bottle by using a 100 mu L sealed glass syringe; then sequentially adding 150-250 mu L of 10mg/mL phosphatidylcholine, 30-50 mu L of 10mg/mL phosphatidylethanolamine and 10-30 mu L of 11mg/mL cholesterol into the glass bottle; volatilizing the organic solvents chloroform and methanol with nitrogen until the formation of thin film lipids at the bottom of the vial was observed; placing the glass bottle in a vacuum drier for at least 30 minutes to remove residual organic solvent; then, 1mL of PBS buffer containing 0.05 to 5pmol of miRNA imic dissolved therein and 10 to 30. mu.L of RNase inhibitor at 11mg/mL were added to the glass vial. The liposome with the similar vesicle particle size can be obtained by a filter or a filter membrane with the pore diameter of 50 nm-800 nm.
2. Removal of free miRNA mimic: transferring the obtained liposome into gel exclusion chromatographic column with pore diameter of 70nm for exclusion chromatographic separation, and collecting components with diameter of 30-1000 nm.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
The molecular weights of phosphatidylcholine, phosphatidylethanolamine and cholesterol used in the examples below were 758.06Da, 907.13Da and 386.65Da, respectively.
"Phosphatidylcholine" used in the examples below was obtained from SIGMA; "phosphatidylethanolamine" is available from SIGMA; "Cholesterol" was purchased from SIGMA; "RNase inhibitor" was purchased from Tiangen, cat # DP 418; "miRNA mimic" was purchased from Ribo organisms.
Example 1 preparation of artificial liposomes containing miRNA mimic (fluorescent liposomes)
1. Method for preparing liposome by constant pressure control extrusion
Add 50. mu.L of chloroform to a 20mL glass vial using a 250. mu.L sealed glass syringe; then 15. mu.L of methanol was added to the same glass bottle using a 100. mu.L sealed glass syringe; then, 108 mu L of 10mg/mL phosphatidylcholine, 21 mu L of 10mg/mL phosphatidylethanolamine and 10 mu L of 11mg/mL cholesterol are sequentially added into the glass bottle; volatilizing the organic solvents chloroform and methanol with nitrogen until the formation of thin film lipids at the bottom of the vial was observed; placing the glass bottle in a vacuum drier for at least 30 minutes to remove residual organic solvent; then, 1mL of a PBS buffer solution (containing mimic 1: 50 fmol; mimic 2: 500 fmol; and mimic 3: 5pmol) containing miRNA mimic was added to the glass vial, and 20. mu.L of an RNase inhibitor was added at 11 mg/mL. The sequence information is as follows: mimic-1: 5'-GGGUACCAUACCGGUUGUCUUA-3', respectively; mimic-2: 5'-UGCUACUCCGAUCUUUAGCCUC-3', respectively; mimic-3: 5'-GUCCCACUCCGUAGAUCUGUUC-3' are provided. The liposome with the similar vesicle particle size can be obtained by a filter or a filter membrane with the pore diameter of 50 nm-800 nm.
2. Removal of miRNA micic free outside liposomes
Removal of free miRNA mimic: transferring the obtained liposome into gel exclusion chromatographic column with pore diameter of 70nm for exclusion chromatographic separation, and collecting components with diameter of 30-1000 nm.
Example 2 identification of artificial liposomes containing miRNA mimic
Take 10. mu.L of the liposome prepared in example 1, and add dd H2O is as follows: diluting by 10 volume ratio; transferring the diluted liquid to carbon film for adsorption for 10min, and adding DI water (deionization)Seed water) cleaning, uranyl acetate UO2(CH3COO)2·2H2O, visual examination of the liposome morphology after 1 minute of staining was performed with a transmission electron microscope (TEM, model JEOL-JEM1400) and recorded by photography. The results show that the particle size of the obtained liposomes is 30-150nm (FIG. 1, a).
mu.L of the liposome prepared in example 1 was taken, 700. mu.L of QIAzol reagent was added thereto, and RNA was extracted by column adsorption method, and dissolved in 30. mu.L of RNase-free water for PCR detection.
The primer information is as follows:
the reverse transcription reaction system is as follows:
| reagent | Volume of |
| 5×PrimeScript Buffer | 2μl |
| PrimeScript RT Enzyme | 0.5μl |
| Water (W) | 4.5μl |
| Reverse transcription primer | 1μl |
| RNA template | 2μl |
Note: primer mix script and RT Enzyme were from TaKaRa reverse transcription Kit (RT reagent Kit, RR 037A).
The reverse transcription reaction procedure was as follows:
the qPCR reaction system was as follows:
| reagent | 20μl |
| 2×Premix Ex Taq | 10μl |
| Primer mixture (10X) | 2μl |
| cDNA | 3μl |
| ddH2O | 5μl |
Note: premix Ex Taq from TaKaRa qPCR kit (Probe qPCR, RR 390A).
The qPCR reaction procedure was as follows:
the result shows that all three miRNA mimic can be stably detected in the liposome, the CT value is 21.7-27.7, and the abundance ratio of mimic-1, mimic-2 and mimic-3 is about 1 according to the PCR quantitative result: 10: 100, which is matched with the feeding ratio in the vesicle preparation process (figure 1, b).
Example 3 stability analysis of artificial liposomes containing miRNA mimic
The prepared artificial liposome is stored at 4 ℃, samples are taken on days 0, 2, 8, 13, 34 and 45 respectively, 10 mu L of the artificial liposome is taken for RNA extraction and PCR quantitative analysis, and the abundance of different RNAs relative to 0 day is detected at different storage time points. The results show that the liposome RNA has good stability, and even if the liposome RNA is stored for more than 45 days, the change amplitude of the CT value of each RNA subjected to PCR is within 0.4 percent, and the liposome RNA shows good stability (figure 2).
Example 4 application of artificial liposome containing miRNA mimic in quality control analysis of extracellular vesicle miRNA extraction process
And adding 8 mul of the artificial liposome prepared in the embodiment 1 into 600 mul of extracellular vesicle solution, dividing the solution into 3 parts, each 200 mul, extracting RNA by adopting a QIAzol kit, and simultaneously detecting the expression quantities of the miRNA, mir-146, mir-150 and the exogenous miRNA mimic-1, mimic-2 and mimic-3 of the vesicle to be detected by adopting a Realtime-PCR method. When the miRNA mimic expression quantity is not used for correction, the PCR result shows that the difference of the expression quantity of the mir-146 and the mir-150 among three parallel samples reaches 150%, and after the miRNA mimic expression quantity is used for correction, the difference of the mir-146 and the mir-150 among the parallel samples is less than 10%, as shown in FIG. 4. The artificial liposome can be used for the extraction process of miRNA of quality control extracellular vesicles.
Example 5 use of artificial liposomes containing miRNA mimic for quantitative analysis of plasma extracellular vesicle miRNA in patient samples
2ml of human plasma is taken, 8 mu L of the artificial liposome prepared in the example 1 is added, after being uniformly mixed, the mixture is divided into three volume gradients of 0.2ml, 0.5ml and 1ml, a gel exclusion chromatographic column is used for carrying out extracellular vesicle separation, and components with the diameter of 30-150nm are collected, and after RNA is extracted. One sample was divided into three equal parts after liposome addition (in order to simulate different recovery rates due to different factors possibly introduced during extraction of vesicle RNA). And simultaneously detecting the expression quantities of the vesicle miRNA, mir-146, mir-150 and the exogenous miRNA mimic-1, mimic-2 and mimic-3 to be detected by adopting a Realtime-PCR method. When the miRNA mimic expression quantity is not used for correction, the PCR result shows that the highest value and the lowest value of the expression quantity obtained by the mir-146 and the mir-150 under different volume separation conditions have a difference of 340 percent at most, and after the miRNA mimic expression quantity is used for correction, the difference between the highest value and the lowest value of the expression quantity of the mir-146 and the mir-150 under different volume separation conditions does not exceed 20 percent, as shown in FIG. 5. The artificial liposome can be used for abundance analysis of sample vesicle miRNA.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (10)
1. The artificial liposome containing miRNA mici is characterized by being formed by mixing a liposome with a lipid bilayer structure and miRNA mici;
the miRNA micic is artificially synthesized non-human miRNA and derivatives thereof.
2. The artificial liposome of claim 1, wherein the raw material for preparing the liposome having a lipid bilayer structure is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, cholesterol, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin.
3. The artificial liposome of claim 2, wherein the molar ratio of phosphatidylcholine to phosphatidylethanolamine to cholesterol in the liposome having a lipid bilayer structure is 5-8:1-3: 1-3.
4. The artificial liposome of claim 1, wherein the derivative of the non-human miRNA is at least one of the following a) -c):
a) the derivative is obtained by replacing part of the base of miRNA with artificial nucleotide base;
b) transforming the framework of miRNA into a phosphorothioate framework to obtain a derivative;
c) and modifying miRNA by polyethylene glycol to obtain the derivative.
5. The artificial liposome according to any one of claims 1 to 4, wherein the artificial liposome has a particle size of 30 to 1000nm and an average density of 1.06 to 1.23 g/mL.
6. The preparation method of the artificial liposome containing miRNA micic is characterized by comprising the following steps:
1) liposomes were prepared by a constant pressure controlled extrusion method: sequentially adding 50-150 mu L of chloroform and 20-50 mu L of methanol into a glass bottle, and then sequentially adding 150-250 mu L of 10mg/mL phosphatidylcholine, 30-50 mu L of 10mg/mL phosphatidylethanolamine and 10-30 mu L of 11mg/mL cholesterol into the glass bottle; volatilizing the organic solvents chloroform and methanol with nitrogen until the formation of thin film lipids at the bottom of the vial is observed; placing the glass bottle in a vacuum drier to remove residual organic solvent; then adding 1mL of PBS buffer solution containing 0.05-5pmol of miRNA imic and 10-30 μ L of RNase inhibitor at 11mg/mL into a glass bottle; obtaining liposome with similar vesicle particle size through a filter or a filter membrane with the pore diameter of 50 nm-800 nm;
2) removal of free miRNA mimic: transferring the liposome obtained in the step 1) to a gel exclusion chromatographic column for column chromatography, and collecting fractions with the particle size of 30-1000 nm;
3) concentration of artificial liposomes: concentrating the fraction collected by column chromatography by ultracentrifugation or ultrafiltration;
wherein the miRNA imic is artificially synthesized non-human miRNA and derivatives thereof;
the derivative of the non-human miRNA is at least one of the following a) to c):
a) the derivative is obtained by replacing part of the base of miRNA with artificial nucleotide base;
b) transforming the framework of miRNA into a phosphorothioate framework to obtain a derivative;
c) and modifying miRNA by polyethylene glycol to obtain the derivative.
7. Use of an artificial liposome according to any of claims 1-5 or prepared according to the method of claim 6 for any of the following applications:
(1) used for evaluating the extraction efficiency of the extracellular vesicle miRNA;
(2) the method is used for quantitative analysis of the miRNA of the extracellular vesicles.
8. A method for evaluating extraction efficiency of extracellular vesicle mirnas, the method comprising: mixing 50-200 mu L of extracellular vesicle extraction sample with 4-20 mu L of the artificial liposome of any one of claims 1-5 or the artificial liposome prepared by the method of claim 6 with known concentration, separating the vesicles by a specific method, extracting RNA in the vesicles, and detecting the expression level of miRNA mimic by a PCR method, thus obtaining the miRNA extraction efficiency of the extracellular vesicle extraction sample.
9. A method for the quantitative analysis of extracellular vesicle mirnas, the method comprising: adding 4-20 mul of artificial liposome into every 1mL of plasma sample, or adding 4-40 mul of artificial liposome into every 20mL of urine sample, or adding 4-40 μ L of artificial liposome into every 2mL of pleural effusion sample, or adding 2-40 μ L of artificial liposome into every 1mL of saliva sample, or adding 2-40 μ L of artificial liposome per 2mL of milk sample, mixing the sample with known concentration of artificial liposome of any one of claims 1-5 or prepared by the method of claim 6, separating the vesicle by specific method, then extracting RNA in the vesicle, respectively detecting the expression quantity of miRNA mimic and endogenous target miRNA by a PCR method, and then comparing the expression quantity of the obtained target miRNA with the expression quantity of the miRNA mimic to obtain the relative expression quantity of the vesicular miRNA in the sample to be detected.
10. The method according to claim 8 or 9, wherein the specific method is ultracentrifugation, gradient density centrifugation, membrane affinity method, polymer precipitation or chromatography; and/or
The method for extracting vesicle RNA is isopropanol precipitation method, ethanol precipitation method or adsorption column method.
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