A method and kit for extracting plasma exosome miRNA

Abstract

The present invention discloses a method and kit for extracting plasma exosome miRNA. The method comprises: using an RNA extraction reagent and a coprecipitant to co-lyze exosomes, mixing with an extractant after lysis, incubating, and centrifuging to obtain a supernatant rich in exosome miRNA, using magnetic beads or a silica gel membrane adsorption column to enrich exosome miRNA, and eluting to obtain plasma exosome miRNA. The present invention can meet the research needs of the development and clinical transformation of plasma exosome miRNA biomarkers, and at the same time, it is low-cost and convenient for large-scale clinical sample processing when used in combination with an automated nucleic acid extraction instrument, saving time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method and a kit for extracting plasma exosome miRNA. Background Art

[0002] Small extracellular vesicles (sEVs) are membrane vesicles with a diameter of about 30-150nm and wrapped in lipid bilayers released by cells, also known as exosomes. The body can continuously secrete exosomes under normal physiological or pathological conditions, and exosomes are widely distributed in various body fluids such as peripheral blood, urine and saliva. With the development of technologies such as second-generation sequencing, proteomics and lipidomics, the rich contents of exosomes have aroused the interest of researchers and have explored the clinical application value of exosomes in early screening and diagnosis of tumors, prognosis monitoring and efficacy prediction. At present, the research on exosomal miRNA is the most. Researchers found that plasma exosome miR-584-5p / miR-106-3p and miR-628-3p / miR-941 can be used for the differential diagnosis of hepatocellular carcinoma, and the sensitivity and specificity of the diagnostic model are 94.1% and 68.4%, respectively (PMID: 33133386). However, for the subsequent clinical transformation of plasma exosome miRNA biomarkers, the selection and cost of plasma exosome miRNA extraction methods need to be focused on and addressed.

[0003] The methods for RNA extraction and purification mainly include solvent extraction, polyacrylamide gel electrophoresis, ultracentrifugation, liquid chromatography, etc. Among them, guanidine isothiocyanate-phenol-chloroform solvent extraction is a more commonly used method, but this method will be contaminated by proteins, salts, DNA, organic solvents and ethanol. Methods based on magnetic beads, ion exchange resins, silica membrane adsorption columns, etc. have also been reported for the extraction of plasma, cell or tissue RNA. Commercial kits can enrich small RNA fragments with a nucleic acid length of less than 200nt by optimizing the adsorption column and experimental system. Among them, Qiagen's kit suitable for serum plasma miRNA extraction (217184, QIAGEN, Shanghai, China) is often used in the study of exosome miRNA-seq. However, the current miRNA extraction kit is relatively expensive, ranging from about 30 to 80 yuan, and it is difficult to use it to process a large number of clinical samples at the same time, and there is a lack of kits and experimental methods for plasma exosome miRNA extraction.

[0004] In summary, most of the currently commercialized miRNA extraction kits are designed for plasma, cells or tissues, and the total RNA extraction amount is at the μg or mg level, while the total RNA content of each 1mL plasma exosome is only about 1-10ng. The cost of commercialized kits is high and they cannot process a large number of clinical samples at the same time. Solvent extraction costs are not high but are prone to introduce contamination such as protein, salt, DNA and organic solvents. Therefore, it is urgent to provide a kit and method that is simple to operate, low-cost and suitable for plasma exosome miRNA extraction. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method and a kit for extracting plasma exosome miRNA, which can meet the research needs of the development and clinical transformation of plasma exosome miRNA biomarkers, and at the same time, has low cost. When used in combination with an automated nucleic acid extraction instrument, it is convenient for large-scale clinical sample processing and saves time.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for extracting plasma exosome miRNA, the method comprising: using an RNA extraction reagent and a co-precipitant to jointly lyse exosomes, mixing with an extractant after lysis, incubating, and centrifuging to obtain a supernatant rich in exosome miRNA, using magnetic beads or silica membrane adsorption columns to enrich exosome miRNA, and eluting to obtain plasma exosome miRNA.

[0008] In the present invention, in response to the need to extract plasma exosome miRNA, an extraction scheme is designed specifically, including using healthy human plasma as a research sample, deeply analyzing the double-layer membrane structure of exosomes, low miRNA content and other characteristics, and establishing a method suitable for plasma exosome miRNA extraction based on magnetic beads or adsorption column filter membrane combined with exosome miRNA. The separation and enrichment efficiency is significantly better than the existing miRNA extraction reagents, which can meet the research needs of the development and clinical transformation of plasma exosome miRNA biomarkers, and at the same time, it is low-cost and convenient for large-scale clinical sample processing when used in combination with an automated nucleic acid extraction instrument, saving time.

[0009] Preferably, the RNA extraction reagent includes: Trizol lysis solution.

[0010] Preferably, the silica gel membrane adsorption column has 8 membrane layers.

[0011] Preferably, the pH of the Trizol lysate solution is 4-6.

[0012] The specific point values ​​in the above 4-6 can be selected as 4, 5, 6, etc.

[0013] Preferably, the RNA extraction reagent comprises TRNzol Universal total RNA extraction reagent.

[0014] In the present invention, the TRNzol Universal total RNA extraction reagent has a stronger ability to lyse exosomes and release miRNA more efficiently.

[0015] Preferably, the coprecipitant comprises: any one of carrier RNA, linear polyacrylamide, glycogen or dextran, or a combination of at least two thereof.

[0016] Preferably, the coprecipitating agent comprises a combination of linear polyacrylamide and glycogen.

[0017] In the present invention, linear polyacrylamide and glycogen are used in synergy to further improve the enrichment effect of trace RNA.

[0018] Preferably, the carrier RNA comprises polyA RNA and / or yeast tRNA.

[0019] Preferably, the amount of the RNA extraction reagent used is 4-10 times the volume of exosomes.

[0020] The specific point values ​​among the above 4-10 times can be selected as 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0021] Preferably, the extractant comprises chloroform or 1-bromo-3-chloropropane.

[0022] Preferably, the amount of the extractant used is 1 / 5-1 / 10 times the volume of the mixed solution after adding the RNA extraction reagent and the coprecipitant.

[0023] The specific point values ​​among the above 1 / 5-1 / 10 can be selected as 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, etc.

[0024] Preferably, the incubation time is 3-7 min.

[0025] The specific point values ​​in the above 3-7min can be selected as 3min, 4min, 5min, 6min, 7min, etc.

[0026] Preferably, the magnetic beads include silanol magnetic beads.

[0027] Preferably, the amount of the magnetic beads used is 1 / 25-1 / 50 of the volume of the supernatant enriched in exosomal miRNA.

[0028] The specific point values ​​among the above 1 / 25-1 / 50 can be selected as 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, etc.

[0029] Preferably, the silica gel membrane adsorption column comprises 6-10 layers of silica gel membrane adsorption columns.

[0030] The specific point values ​​in the above 6-10 layers can be selected from 6th layer, 7th layer, 8th layer, 9th layer, 10th layer, etc.

[0031] Preferably, the method for extracting plasma exosome miRNA comprises the following steps:

[0032] (1) Add 10 ng / μL-30 ng / μL of coprecipitant to the exosomes and mix well, then add 4-10 times the volume of RNA extraction reagent and mix well, incubate at 20-30°C for 3-7 min, add 1 / 5-1 / 10 times the volume of extraction agent and mix well, incubate at 20-30°C for 3-7 min, centrifuge after the incubation, and take the upper aqueous phase;

[0033] (2) mixing the aqueous phase with magnetic beads and adding isopropanol to promote the binding of miRNA to the magnetic beads, adding a washing solution for washing, discarding the supernatant, adding water for incubation, taking the supernatant, and obtaining miRNA; or,

[0034] The aqueous phase is mixed with anhydrous ethanol to reduce the solubility of miRNA in the solution, and the mixture is placed in a silica membrane adsorption column. The solution is discarded by centrifugation, and detergent is continuously added to the silica membrane adsorption column. The solution is discarded by centrifugation. The washing is repeated 1 to 3 times. Water is added to the silica membrane adsorption column. The solution is collected by centrifugation to obtain miRNA.

[0035] Preferably, the detergent comprises an ethanol solution.

[0036] Preferably, the volume percentage of ethanol in the ethanol solution is 70%-90%.

[0037] The specific point values ​​among the above 70%-90% may be 70%, 72%, 74%, 76%, 80%, 82%, 84%, 86%, 90% and the like.

[0038] In a second aspect, the present invention provides a kit for extracting plasma exosome miRNA, the kit comprising an RNA extraction reagent, a coprecipitant, an extractant, a detergent, an eluent and magnetic beads.

[0039] Preferably, the RNA extraction reagent includes: Trizol lysis solution.

[0040] Preferably, the pH of the Trizol lysis solution is 4-6.

[0041] Preferably, the coprecipitant comprises: any one of carrier RNA, linear polyacrylamide, glycogen or dextran, or a combination of at least two thereof.

[0042] Preferably, the extractant comprises: chloroform or 1-bromo-3-chloropropane.

[0043] Preferably, the detergent comprises: an ethanol solution.

[0044] Preferably, the eluent comprises: ddH2O free of DNase and RNase.

[0045] Preferably, the magnetic beads include silanol magnetic beads.

[0046] Preferably, the coprecipitant is used at a concentration of 10-30 ng / μL.

[0047] The specific point values ​​among the above 10-30ng / μL can be selected as 10ng / μL, 12ng / μL, 14ng / μL, 20ng / μL, 22ng / μL, 24ng / μL, 26ng / μL, 28ng / μL, 30ng / μL, etc.

[0048] Preferably, the volume percentage of ethanol in the ethanol solution is 70%-90%.

[0049] The specific point values ​​among the above 70%-90% may be 70%, 72%, 74%, 76%, 80%, 82%, 84%, 86%, 90% and the like.

[0050] In a third aspect, the present invention provides use of the method described in the first aspect or the kit described in the second aspect in extracting plasma exosome miRNA.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention develops a method and a kit suitable for extracting plasma exosome miRNA, which can meet the research needs of the development and clinical transformation of plasma exosome miRNA biomarkers, and at the same time, it is low-cost and convenient for large-scale clinical sample processing when used in combination with an automated nucleic acid extraction instrument, saving time;

[0053] (2) The plasma exosome miRNA extraction method established by the present invention has good enrichment efficiency for low GC content miRNA, which can meet the subsequent clinical detection needs of low GC content miRNA;

[0054] (3) The plasma exosome miRNA extraction method established by the present invention is equivalent to or better than the domestic miRNA extraction kit, but the cost of miRNA extraction of the present invention is as low as four yuan, which is one tenth of the domestic miRNA extraction kit;

[0055] (4) The plasma exosome miRNA extraction method established in the present invention is based on the principle of the magnetic bead method and can be used in combination with an automated nucleic acid extraction instrument to facilitate large-scale clinical sample processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the result of transmission electron microscopy identification of plasma exosomes;

[0057] Figure 2 This is the result diagram of plasma exosome particle size distribution. DETAILED DESCRIPTION

[0058] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0059] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0060] Example 1

[0061] In this example, plasma exosome miRNA was extracted.

[0062] (1) Sample collection and grouping

[0063] A total of 6 healthy blood samples were collected. Each person took 1 mL of separated plasma, mixed it in two tubes, and then divided it into three parts for subsequent exosome isolation and miRNA extraction. The experiment was divided into three groups according to the isolation method of exosome miRNA: 3DMed-magnetic bead group, 3DMed-adsorption column group and Bio-bio group, and each group was repeated three times.

[0064] (2) Plasma sample separation

[0065] The blood samples of healthy people used were collected into 10mL anticoagulated vacuum blood collection tubes (REF367525, BD, USA), and then the samples were sent at 4℃. The received blood samples were centrifuged at 1600g for 10min at 4℃ to remove cells or cell debris. After centrifugation, the supernatant was transferred and centrifuged for 15min at 16000g for the second time at 4℃. After centrifugation, the supernatant was transferred to 1.5mL centrifuge tubes with 1mL per tube and stored in a -80℃ refrigerator for use.

[0066] (3) Isolation and identification of plasma exosomes

[0067] The plasma samples were taken out and placed in a 37℃ water bath to thaw, centrifuged at 12000g for 10min at 4℃, and the supernatant of each two tubes of plasma was transferred to a 2mL centrifuge tube. After mixing, it was divided into three portions, each with 600μL of plasma. 150μL of L-type exosome precipitant (L3525, 3DMed, Shanghai, China) was added to each plasma sample, mixed, incubated at 4℃ for 30min, centrifuged at 4700g for 30min at 4℃, and the supernatant was discarded. 80μL PBS (phosphate buffer saline) was added to the 3DMed-magnetic bead group and the 3DMed-adsorption column group, and 200μL PBS was added to the Bio-bio group to resuspend the exosome precipitate.

[0068] Morphological characteristics of plasma exosomes: First, the exosomes were resuspended in PBS, and then 4% paraformaldehyde was added to fix the exosomes. The exosomes were dropped onto a carbon-coated 200-mesh electron microscope copper grid. The copper grid was washed twice with PBS, washed with PBS containing glycine (50 mM) for 3 min, and washed again with PBS containing 0.5% BSA for 10 min. Finally, the copper grid was stained with 2% uranyl acetate. After staining, the exosome morphology was characterized by transmission electron microscopy (H-7650, Hitachi High-Technologies, Japan).

[0069] Detection of plasma exosome particle size distribution: First, the exosomes were diluted to 1×10 9 / mL, pipetting and mixing. Subsequently, the NTA (NanoSight NS300, Malvern, UK) instrument was turned on to inject the diluted sample into the sample chamber, and the 488nm excitation module was used. The lens parameters of the camera were set, the shutter value was selected as 890, the gain value was selected as 146, and the detection threshold was set to 7. At least 200 complete tracks were analyzed and obtained for each video. Finally, the nanoparticle tracking data of exosomes were analyzed using the NTA analysis software (version 2.3).

[0070] (4) Extraction of plasma exosome miRNA

[0071] Isolation of plasma exosome miRNA by 3DMed-magnetic beads

[0072] 3DMed-magnetic bead group separation plasma exosome miRNA extraction kit, including linear polyacrylamide, TRNzolUniversal total RNA extraction reagent (standard reagent), chloroform, silicon hydroxy magnetic microspheres, isopropanol, 80% ethanol and RNase-free water.

[0073] The extraction process using this kit is as follows: add 30 ng / μL linear polyacrylamide (Sigma, 749222, USA) to the resuspended exosomes and mix well, then add 10 times the volume of TRNzol Universal total RNA extraction reagent (TIANGEN, DP424, Shanghai, China) and vortex to mix well, incubate at 25°C for 7 minutes, add 1 / 10 times the volume of chloroform in the mixture and vortex to mix well, and incubate at 25°C for 7 minutes. After the incubation, centrifuge at 12000g for 15 minutes at 4°C. After the centrifugation, transfer the upper aqueous phase to a new centrifuge tube, add 10 μL of silicon hydroxy magnetic microspheres (LNJNBIO, 160024, Shanghai, China) and vortex to mix well, then add 2 times the volume of isopropanol and mix well, and shake at 25°C 2000rpm for 7 minutes. Transfer the sample to the magnetic rack for 5 minutes, and discard the supernatant after the solution becomes clear. Add 1mL of freshly prepared 80% ethanol, shake and centrifuge at 25℃ for 30 seconds, transfer the sample to a magnetic rack for 5min, and discard the supernatant after the solution becomes clear. Repeat the wash once, discard the supernatant, open the tube cap and dry for 5min. Add 15μL RNase-free water to the sample tube, shake at 25℃, 2000rpm for 7min. Transfer the sample to a magnetic rack for 5min, and aspirate the supernatant into a new 1.5mL centrifuge tube after the solution becomes clear.

[0074] Example 2

[0075] This example is used to extract plasma exosome miRNA, which is different from Example 1 in that the process of step (4) is: 3DMed-adsorption column group separates plasma exosome miRNA.

[0076] The 3DMed-adsorption column group separation plasma exosome miRNA extraction kit includes linear polyacrylamide, TRNzolUniversal total RNA extraction reagent (standard reagent), chloroform, 100% ethanol, adsorption column, 80% ethanol and RNase-free water.

[0077] The extraction process using this kit is as follows: add 30 ng / μL linear polyacrylamide (Sigma, 749222, USA) to the resuspended exosomes and mix well, then add 10 times the volume of TRNzol Universal total RNA extraction reagent (TIANGEN, DP424, Shanghai, China) and vortex to mix well, incubate at 25°C for 7 minutes, add 1 / 10 times the volume of chloroform in the mixture and vortex to mix well, and incubate at 25°C for 7 minutes. After the incubation, centrifuge at 12000g for 15 minutes at 4°C. After the centrifugation, transfer the upper aqueous phase to a new centrifuge tube, add 1.5 times the volume of 100% ethanol, and mix thoroughly. Pipette 700μL of the mixture into an 8-layer silica gel membrane adsorption column (Supin, Nantong, China), centrifuge at 8000g at 25°C for 15 seconds, and discard the centrifuged waste until all the mixture passes through the silica gel membrane adsorption column. Add 700 μL of freshly prepared 80% ethanol, centrifuge at 8000g for 15 seconds at 25℃, and discard the waste liquid. Add 500 μL of 80% ethanol, centrifuge at 8000g for 15 seconds at 25℃, and discard the waste liquid. Add 500 μL of 80% alcohol, centrifuge at 8000g for 2 minutes at 25℃, discard the waste liquid, transfer the silica gel adsorption column to a new collection tube, and centrifuge at 20000g for 5 minutes at 25℃. Transfer the silica gel adsorption column to a new 1.5mL centrifuge tube, add 15 μL of RNase-free water to the silica gel adsorption column, incubate at 25℃ for 1 minute, and then centrifuge at 25℃ for 1 minute at 20000g, collect the miRNA sample and repeat the elution.

[0078] Example 3

[0079] This example is used to extract plasma exosome miRNA. The only difference from Example 1 is that linear polyacrylamide is replaced with an equal amount of carrier RNA (carrier RNA, 19701ES03, Yisheng Biotechnology).

[0080] Example 4

[0081] In this example, plasma exosome miRNA was extracted. The only difference from Example 1 was that linear polyacrylamide was replaced with an equal amount of glycogen (glycogen, D0818, Bio-Tech; AM9510).

[0082] Example 5

[0083] This example is used to extract plasma exosome miRNA. The only difference from Example 1 is that linear polyacrylamide is replaced with an equal amount of dextran (9004-54-0, Merck).

[0084] Example 6

[0085] This example is used to extract plasma exosome miRNA. The only difference from Example 1 is that the linear polyacrylamide is replaced with 15 ng / μL linear polyacrylamide and 15 ng / μL glycogen.

[0086] Example 7

[0087] This example is used to extract plasma exosome miRNA, and the only difference from Example 1 is that the linear polyacrylamide is replaced with 15 ng / μL linear polyacrylamide and 15 ng / μL carrier RNA.

[0088] Example 8

[0089] This example is used to extract plasma exosome miRNA. The only difference from Example 1 is that the linear polyacrylamide is replaced with 15 ng / μL glycogen and 15 ng / μL carrier RNA.

[0090] Comparative Example 1

[0091] The difference from Example 1 is that the process of step (4) is as follows: Bio-bio isolates plasma exosome miRNA.

[0092] BIOG plasma serum miRNA extraction kit (BAIDAI, 51075, Shanghai, China) was used to extract plasma exosome miRNA. The specific steps are as follows: add 200 μL miRNA releaser and 20 μL digestion solution to the resuspended exosomes, vortex and mix, and place in a 65°C water bath for digestion for 10 min. Add 5 μL miRNA enrichment agent and 850 μL anhydrous ethanol, mix by inversion, transfer 650 μL of the mixture to the centrifuge column, centrifuge at 12000 rpm at 25°C for 1 min, and discard the waste liquid from the centrifuge until all the mixture passes through the centrifuge column. Add 500 μL washing solution A, let it stand at 25°C for 2 min, centrifuge at 12000 rpm at 25°C for 1 min, and discard the waste liquid from the centrifuge. Add 500 μL washing solution B, let it stand at 25°C for 2 min, centrifuge at 12000 rpm at 25°C for 1 min, and discard the waste liquid from the centrifuge. Add 500 μL of washing solution B, centrifuge at 12000 rpm at 25℃ for 1 min, discard the waste liquid, and centrifuge again at 12000 rpm at 25℃ for 2 min. Transfer the centrifuge column to a new 1.5 mL centrifuge tube, open the lid and place at 25℃ for 2 min, add 15 μL of elution solution preheated at 65℃ to the centrifuge column membrane, incubate at 25℃ for 2 min, and centrifuge at 25℃ at 12000 rpm for 1 min to collect miRNA samples.

[0093] Test Example 1

[0094] In order to verify the quality of the isolated plasma exosomes, taking Example 1 as an example, the morphology of plasma exosomes was detected by transmission electron microscopy. Figure 1As shown in Figure 2, the extracted exosomes showed a typical "horseshoe-shaped" morphology of exosomes. The particle size distribution of plasma exosomes was detected by NTA. The results were as follows: Figure 2 As shown, the main peak of the extracted exosome particle size is 116 nm, which is consistent with the particle size distribution of exosomes.

[0095] Test Example 2

[0096] Detection of plasma exosome miRNA content.

[0097] In order to verify the effect of the plasma exosome miRNA extraction method established by the present invention, we selected the relative expression of miRNAs with high, medium and low GC contents to evaluate the enrichment efficiency of miRNAs with different GC contents. Among them, miRNAs with low GC contents refer to miRNAs with GC contents ≤ 30%, miRNAs with medium GC contents refer to miRNAs with GC contents between 30% and 60%, and miRNAs with high GC contents refer to miRNAs with GC contents ≥ 60%. Through literature search for potential exosome miRNA markers and calculation of the GC content of miRNAs, four miRNAs, hsa-miR-340-5p (GC content is 27.3%), hsa-miR-452-3p (GC content is 40.9%), hsa-miR-99a-5p (GC content is 50.0%) and hsa-miR-423-3p (GC content is 65.2%), were selected as representatives for detection.

[0098] The relative expression of exosomal miRNA was detected by qPCR. Exosomal miRNA was synthesized using TaqMan advancedmiRNA cDNA synthesis kit (Applied Biosystems TM , A28007, USA) for reverse transcription of exosomal miRNA, followed by TaqMan TM Fast Advanced Master Mix (Applied Biosystems TM ,4444557, USA) and TaqMan Advanced miRNA Assay (Applied Biosystems TM ,A25576,USA) to configure the qPCR reaction system, and finally ABI 7500 was used for fluorescence quantification. The specific experimental process refers to the product manual.

[0099] The results of fluorescence signal detection of miRNAs with high, medium and low GC contents are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] Results: The 3DMed-magnetic bead group and 3DMed-adsorption column group of the plasma exosome miRNA extraction method established in Examples 1-8 could detect the fluorescence signals of hsa-miR-340-5p, hsa-miR-452-3p, hsa-miR-99a-5p and hsa-miR-423-3p within 40 cycles; in the Biotech Biotech kit group of Comparative Example 1, except for hsa-miR-452-3p, no fluorescence signals were detected for the other three miRNAs.

[0104] Example 1 detected CT values ​​of 4 miRNAs and the CT value was <36 cycles, while the CT of hsa-miR-452-3p detected by the EMI Bio Group in Comparative Example 1 was >36 cycles, indicating that the amount of miRNA extracted was low, and compared with Example 1, the CT value results of hsa-miR-340-5p, hsa-miR-99a-5p and hsa-miR-423-3p in Example 3 were similar, but the expression level of hsa-miR-452-3p was reduced by 3 times. Example 6 detected that the CT values ​​of the 4 miRNAs were all lower, that is, amplification required fewer cycles, indicating that the amount of extracted miRNA was more, indicating that the present invention uses linear polyacrylamide and glycogen to cooperate, which can further promote the enrichment of exosomal miRNA, while Examples 7 and 8 detected that the CT values ​​of the 4 miRNAs were higher, that is, amplification required more cycles, indicating that the amount of extracted miRNA was less, indicating that linear polyacrylamide and carrier RNA or glycogen and carrier RNA cannot cooperate.

[0105] In summary, the present invention designs an extraction scheme for plasma exosome miRNA, designs a specific extraction process and cooperates with specific reagents, which can effectively extract and enrich miRNA in plasma exosomes. In addition, it has good enrichment efficiency for miRNA with low GC content, and can also meet the subsequent clinical detection needs of low GC content miRNA. At the same time, it is low-cost and can meet the research needs of the development and clinical transformation of plasma exosome miRNA biomarkers. It can also be used in combination with an automated nucleic acid extraction instrument to facilitate large-scale clinical sample processing and save time.

[0106] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for extracting plasma exosome miRNA, characterized in that: The method comprises: Exosomes were lysed together with RNA extraction reagent and co-precipitant, and then mixed with extraction agent, incubated and centrifuged to obtain supernatant rich in exosomal miRNA. Exosomal miRNA was enriched using magnetic beads or silica membrane adsorption columns, and plasma exosomal miRNA was eluted.

2. The method according to claim 1, characterized in that The RNA extraction reagent includes: Trizol lysis solution; Preferably, the pH of the Trizol lysis solution is 4-6.

3. The method according to claim 1 or 2, characterized in that: The coprecipitant comprises: any one or a combination of at least two of carrier RNA, linear polyacrylamide, glycogen or dextran; Preferably, the coprecipitating agent comprises a combination of linear polyacrylamide and glycogen.

4. The method according to claim 3, characterized in that The carrier RNA includes polyA RNA and / or yeast tRNA.

5. The method according to any one of claims 1 to 4, characterized in that The dosage of the RNA extraction reagent is 4-10 times the volume of exosomes.

6. The method according to any one of claims 1 to 5, characterized in that The extractant includes chloroform or 1-bromo-3-chloropropane; Preferably, the amount of the extractant is 1 / 5-1 / 10 times the volume of the mixed solution after adding the RNA extraction reagent and the coprecipitant; Preferably, the incubation time is 3-7 min.

7. The method according to claim 1, characterized in that The magnetic beads include silicon hydroxy magnetic beads; Preferably, the amount of the magnetic beads used is 1 / 25-1 / 50 volume of the supernatant rich in exosomal miRNA; Preferably, the silica gel membrane adsorption column comprises 6-10 layers of silica gel membrane adsorption columns.

8. A kit for extracting plasma exosome miRNA, characterized in that: The kit comprises an RNA extraction reagent, a coprecipitant, an extractant, a detergent, an eluent and magnetic beads; Preferably, the RNA extraction reagent comprises: Trizol lysis solution; Preferably, the pH of the Trizol lysis solution is 4-6; Preferably, the coprecipitant comprises: any one or a combination of at least two of carrier RNA, linear polyacrylamide, glycogen or dextran; Preferably, the coprecipitant is a combination of linear polyacrylamide and glycogen; Preferably, the extractant comprises: chloroform or 1-bromo-3-chloropropane; Preferably, the detergent comprises: an ethanol solution; Preferably, the eluent comprises: ddH2O free of DNase and RNase; Preferably, the magnetic beads include silanol magnetic beads.

9. The kit according to claim 8, characterized in that The concentration of the coprecipitant is 10-30 ng / μL; Preferably, the volume percentage of ethanol in the ethanol solution is 70%-90%.

10. Use of the method according to any one of claims 1 to 7 or the kit according to claim 8 or 9 in extracting plasma exosome miRNA.

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