CN105368931A - Method for detecting exosome-derived miRNAs biomarkers in urine - Google Patents
Method for detecting exosome-derived miRNAs biomarkers in urine Download PDFInfo
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- CN105368931A CN105368931A CN201510664323.5A CN201510664323A CN105368931A CN 105368931 A CN105368931 A CN 105368931A CN 201510664323 A CN201510664323 A CN 201510664323A CN 105368931 A CN105368931 A CN 105368931A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a method for detecting exosome-derived miRNAs biomarkers in urine, and belongs to the field of DNA or RNA separating and preparing or purifying methods. The method includes the steps that exosomes are extracted from the urine of people, and then subjected to morphology and protein identification through an electron microscope and a Western blot, and size distribution analysis is conducted on the exosomes through NanoSight LM10; total RNA containing miRNAs is extracted from the exosomes after identification and analysis, and purification analysis is conducted on the total RNA through a freeze-drying method; afterwards, inverse transcription and fluorogenic quantitative PCR amplification are carried out, and the needed miRNAs biomarkers are separated and identified. According to the method, the urine is used as an experimental material, and the advantages of being easy and convenient to collect, noninvasive and large in number are achieved; a large quantity of exosomes are contained in the urine, hence, the material basis is provided for identification of the miRNAs biomarkers, and wide application prospects are achieved.
Description
Technical field
The invention belongs to separation, preparation or the method field of purify DNA or RNA, specifically the detection method of the miRNAs biomarker of exosome in a kind of urine.
Background technology
Exosome is the chamber intracellular vesicle of many vesicas body (multivesicularbodies, MVBs) in cell, is released in biological fluid in a large number, the body fluid such as such as blood, urine, cerebrospinal fluid, pernicious ascites pleural fluid, amniotic fluid and synovia.Be released in extracellular environment in the mode of exocytosis after MVBs and cell membrane fusion, diameter is 30 ~ 150nm.Exosome contains multiple proteins, mRNAs and miRNAs, exosome can carry RNA molecule and be circulated in blood the cell-cell communication carrying out growing distance.
At present, be serum and blood plasma with the common biological fluid that non invasive method is collected, wherein circulation miRNAs receives much concern in recent years.Compared with mRNAs, circulation miRNAs is more stable, can tolerate storage and freeze-thaw cycle, reduces mechanical degradation, also can resist the biochemical degradations such as rnase in serum (RNAse) and external RNAseA simultaneously.In the blood environment being imbued with RNAse, many mechanism protection circulation miRNAs, wherein exosome is considered to a kind of main protection mechanism.The adventitia of exosome provides comprehensive protection to RNA, itself and the extracellular environment being easy to digest is completely cut off.
Be urine with the common biological fluid of another kind that non invasive method is collected, urine not only easily obtains, and collect easy, without wound, quantity is large.The miRNAs be present in urine is roughly the same with the protection mechanism of circulation miRNAs; but because urinary fractions is complicated; and it is active to there is a large amount of RNAse in urinary system, make the naked miRNAs in urine be easier to degraded, therefore study the miRNAs coming from exosome and have more realistic meaning.
Summary of the invention
The present invention derives from the preparation method of the exosome of urine and therefrom extracts and identify the method for the miRNAs biomarker that it comprises to provide a kind of, the detection method of exosome source property miRNAs biomarker in a kind of urine proposed.
The present invention realizes according to following technical scheme.
A detection method for the miRNAs biomarker of exosome in urine, comprises the following steps:
The preparation of a.exosome
Collecting urine, is urine according to volume ratio: the ratio of protease inhibitor cocktail=50:4.22 adds protease inhibitor cocktail; Centrifugal segregation cell precipitation and film fragment under 1-4 ° of C, 1800-2200g condition; Get supernatant, centrifugal segregation major diameter many vesicas body under 1-4 ° of C, 15000-17000g condition; Get supernatant, centrifugal collecting precipitation under 1-4 ° of C, 150000-200000g condition; With without the resuspended precipitation of RNAse water, centrifugal collecting precipitation under 1-4 ° of C, 150000-200000g condition, obtains exosome again;
B.totalRNA extracts and purifying
Be amount of urine according to volume ratio: the ratio of TRIzolLS solution=100:1, in the precipitation that step a obtains, add TRIZOLLS solution, mixing; And be TRIZOLLS solution according to volume ratio: the ratio of chloroform=5:1, adds chloroform, centrifugal; Draw upper strata aqueous phase and add Virahol, hold over night, centrifugal; After 75% washing with alcohol precipitation, centrifugal collecting precipitation, dry, precipitate with without the resuspended RNA of RNAse water; TotalRNA after the above-mentioned dissolving of vacuum freeze drier purifying;
C. miRNAcDNA first chain is synthesized
1. miRNA3' end adds Poly (A) reaction;
2. the miRNA reverse transcription reaction modified of Poly (A);
D. fluorescent quantitation qPCR reacts
By step c 2. in obtain miRNA first chain cDNA carry out hsa-miR-15b-3P, hsa-miR-15a-3P and hsa-miR-135b-5P fluorescent quantitation detect.
Present invention obtains following beneficial effect: the invention provides a kind of derive from the exosome of urine preparation method and the method for wherein the comprised miRNAs biomarker of qualification.With from serum with extract exosome in blood plasma and compared with the method for further enrichment miRNAs, biological fluid used in the present invention more easily obtains, collect easy, large without wound, quantity.Moreover, in urine, there is a large amount of exosome, and the adventitia of exosome has good provide protection to miRNAs wherein.Present invention obtains the kind of the exosome that derives from urine and wherein miRNAs, have broad application prospects.
Accompanying drawing explanation
Fig. 1 is exosome form and size figure under transmission electron microscope of the present invention;
Fig. 2 is that NanoSightLM10 of the present invention detects exosome Analyzing on Size figure;
Fig. 3 is the qualification figure that WesternBlot of the present invention detects exosome labelled protein TSG101 and CD9;
Fig. 4 is that RT-qPCR of the present invention carries out the rear mean CT-number analysis chart of hsa-miR-15b-3P, hsa-miR-15a-3P and hsa-miR-135b-5P detection.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described in detail.
The preparation of 1.exosome: leave and take mud-stream urine 200ml in early morning, every 50ml urine adds protease inhibitor cocktail and (comprises the 100mMNaN of 1.67ml
3, the 1mMLeupeptin of the 10mMPMSF of 2.5ml, 50ul); Centrifugal 10 minutes of 4 ° of C, 2000g, remove cell precipitation and film fragment; Get supernatant 4 ° of C, 17000g centrifugal 45 minutes, remove major diameter many vesicas body; Get supernatant 4 ° of C, 200000g(BECKMANCOULTER, L-100XPUltracentrifuge) centrifugal 65 minutes; Remove supernatant, without the resuspended precipitation of RNAse water; 4 ° of C, 200000g recentrifuge 65 minutes, gained precipitation is exosome;
Every 50ml urine gained precipitation is according to further experiment object, dissolve with the different solutions of same volume, use 30ul anhydrous alcohol solution for electron microscopic observation, that detects for WesternBlot and NanoSightLM10 uses 30ulPBS solubilize, and exosome lysate saves backup in-80 DEG C.
The electron microscopic observation of 2.Exosome and NanoSightLM10 analyze: by anhydrous alcohol solution exosome precipitation, and ultrasonic wave dispersion exosome suspension 5 minutes, by a little hanging drop on ultrathin carbon films copper mesh, fully dry, 1 ‰ phospho-wolframic acid negative staining, fully dry, and transmission electron microscope observing, take pictures.As shown in Figure 1, the exosomes corpusculum in the negative staining electromicroscopic photograph display urine source of exosomes is flat or spherical corpusculum, and diameter is about about 50nm (in Fig. 1 shown in arrow); As shown in Figure 2, the exosome size in NanoSightLM10 analysis display urine of the present invention within 30nm ~ 500nm scope, but mainly concentrates on 140nm place.
The WesternBlot of 3.Exosome detects: dissolve exosome precipitation with 30ulPBS, BCA method measures exosome protein concentration; Calculate protein concentration, add irreducibility 5 × SDS sample-loading buffer, 95 ° of C sex change 5 minutes; The concentrated glue of preparation 5% and 12% separation gel; By mixture loading after sex change, electrophoresis (during concentrated glue, voltage is 80V, and during separation gel, voltage is about 150V); Application 0.22umPVDF film carries out electricity and turns, 100V1 hour; 5% milk closes 10 minutes; Primary antibodie CD9 dilutes 1500 times, and TSG101 dilutes 2000 times, incubated at room 90 minutes; General two anti-(horseradish enzyme labelling goat antirabbit or mouse IgG) dilute 5000 times, incubated at room 45 minutes; Development, takes pictures (developing solution is instant joins).As shown in Figure 3, WesternBlot detects same routine sample exosome marker protein TSG101 and CD9 and all expresses, and TSG101 expresses the expression higher than CD9.
4. comprise extraction and the purifying of the totalRNA of miRNAs: 100ml urine ultracentrifugation gained exosome precipitation adds 1mlTRIzolLS, piping and druming mixing, room temperature places 5 minutes; Add 0.2ml chloroform, add a cover and acutely rock 15 seconds afterwards, room temperature places 3 minutes, 12000g, 4 ° of C, and centrifugal 15 minutes, RNA was present in aqueous phase; By upper water phase transition in another clean centrifuge tube, add 0.5ml Virahol, 1-4 ° of C hold over night, 12000g, 4 ° of C, centrifugal 10 minutes; Remove supernatant, add 1ml75% ethanol (without the preparation of RNase water), washing RNA precipitation, vortex (Shanghai Hu Xi analytical instrument factory, the miniature vortex mixed instrument of WH-2) mixes, 7500g, 4 ° of C, centrifugal 5 minutes; Remove supernatant, put air drying 5 minutes, precipitate, repeatedly blow and beat with without the resuspended RNA of RNase water, 55 ° of C leave standstill 10 minutes; TotalRNA is through vacuum-freeze-dry instrument (NingBo XinZhi Biology Science Co., Ltd, SCIENTZ-10N freeze drier) freeze concentration; Through NanoDropND-2000 spectrophotometric determination concentration and OD value after concentrated, concentration is generally 10 ~ 25ng/ul, OD
260/ OD
280ratio is greater than 1.8.
5.miRNAcDNA first chain synthesizes: application miRcutemiRNAcDNA first chain synthetic agent box (TIANGENBIOTECH (BEIJING) CO., LTD), catalog number (Cat.No.) KP201 carries out the first chain synthesis.
1. miRNA3' end add Poly (A) process: on ice precooling RNaseFree reaction tubes in add following reagent: gained TotalRNA12 μ L in step 4,
e.colipoly (A) Polymerase (5U/ μ L) 0.4 μ L, 10 × Poly (A) PolymeraseBuffer2 μ L, 5 × rATPSolution4 μ L, RNase-FreeddH O1.6 μ L, cumulative volume 20 μ L, finally adds
e.colipoly (A) Polymerase;
2. pipettor mixes the reaction solution of above-mentioned preparation gently, of short duration centrifugal after react 60min at 37 ° of C;
3. the miRNA reverse transcription reaction modified of Poly (A): by step 5 2. in the sample 2 μ L that obtains and 10 × RTPrimer2 μ L, 10 × RTBuffer2 μ L, SuperPuredNTPs (2.5mM) 1 μ L, RNasin (40U/ μ L) 1 μ L, OuantRTase0.5 μ L, RNase-FreeddH O11.5 μ L, totally 20 μ L reaction solutions mix gently, of short duration centrifugal after react 60min at 37 ° of C;
6. fluorescent quantitation qPCR reacts: application miRcutemiRNA fluorescence quantitative detection kit (TIANGENBIOTECH (BEIJING) CO., LTD), catalog number (Cat.No.) FP401, the fluorescent quantitation carrying out hsa-miR-15b-3P, hsa-miR-15a-3P and hsa-miR-135b-5P detects.
1. hsa-miR-15b-3P, the upstream primer of hsa-miR-15a-3P and hsa-miR-135b-5P is designed by TIANGEN Biotech (Beijing) Co., Ltd. and synthesizes, catalog number (Cat.No.) is respectively CD201-0231, CD201-0229 and CD201-0213, and downstream primer is test kit universal primer.
2. reagent is placed on ice, and according to following proportional arrangement reaction system: 2 × miRcutemiRNAPremix (withSYBR & ROX) 10 μ L, ForwardPrimer0.4 μ L (concentration 10 μMs), ReversePrimer0.4 μ L (concentration 10 μMs), step 5 is the middle miRNA first chain cDNA2 μ L obtained 3., RNase-FreeddH O7.2 μ L, totally 20 μ L reaction systems;
3. above-mentioned reaction solution being placed in 48 hole qPCR reacts in instrument (Illumina, IncSanDiego, CA92122USA), 94 ° of C, 2min denaturations; 45 circulations (94 ° of C, 20sec; 62 ° of C, 34sec); 94 ° of C, 20sec, 62 ° of C, 34sec, carry out data processing and chart production in 94 ° of C, 20sec, MicrosoftOfficeExcel.As shown in Figure 4, application RT-qPCR amplifies hsa-miR-15b-3P from the miRNAs that urine exosome comprises, and the Ct value of hsa-miR-15a-3P and hsa-miR-135b-5P tri-kinds of miRNAs is respectively 31.3,29.69 and 31.35, and amplification number of times is 22 times.
Claims (3)
1. the detection method of exosome source property miRNAs biomarker in urine, is characterized in that: comprise the following steps:
The preparation of a.exosome
Collecting urine, is urine according to volume ratio: the ratio of protease inhibitor cocktail=50:4.22 adds protease inhibitor cocktail; Centrifugal segregation cell precipitation and film fragment under 1800-2200g condition; Get supernatant, centrifugal segregation major diameter many vesicas body under 15000-17000g condition; Get supernatant, centrifugal collecting precipitation under 150000-200000g condition; With without the resuspended precipitation of RNAse water, centrifugal collecting precipitation under 150000-200000g condition, obtains exosome again; Above centrifugally operated all carries out under 1-4 ° of C condition;
B.totalRNA extracts and purifying
Be amount of urine according to volume ratio: the ratio of TRIzolLS solution=100:1, in the precipitation that step a obtains, add TRIZOLLS solution, mixing; And be TRIZOLLS solution according to volume ratio: the ratio of chloroform=5:1, adds chloroform, centrifugal; Draw upper strata aqueous phase and add Virahol, hold over night, centrifugal; After 75% washing with alcohol precipitation, centrifugal collecting precipitation, dry, precipitate with without the resuspended RNA of RNAse water; TotalRNA after the above-mentioned dissolving of vacuum freeze drier purifying;
C. miRNAcDNA first chain is synthesized
1. miRNA3' end adds Poly (A) reaction;
2. the miRNA reverse transcription reaction modified of Poly (A);
D. fluorescent quantitation qPCR reacts
By step c 2. in obtain miRNA first chain cDNA carry out hsa-miR-15b-3P, hsa-miR-15a-3P and hsa-miR-135b-5P fluorescent quantitation detect.
2. the detection method of exosome source property miRNAs biomarker in a kind of urine according to claim 1, is characterized in that: the centrifugation time removing cell precipitation and film fragment is 10-15min; The centrifugation time removing major diameter many vesicas body is 45-50min; The centrifugation time of collecting precipitation is 60-65min.
3. the detection method of exosome source property miRNAs biomarker in a kind of urine according to claim 1, is characterized in that: the composition of described protease inhibitor cocktail is 100mMNaN
3, 10mMPMSF, 1mMLeupeptin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107271655A (en) * | 2017-05-18 | 2017-10-20 | 成都中医药大学附属医院 | A kind of kit and method for detecting urine excretion body load miRNAs |
| CN110964694A (en) * | 2019-11-11 | 2020-04-07 | 浙江卫未生物医药科技有限公司 | Method for extracting exosome based on density gradient centrifugation and ultracentrifugation |
| CN112126643A (en) * | 2020-09-11 | 2020-12-25 | 上海长征医院 | Method for separating ecDNA (deoxyribose nucleic acid) in exosome based on magnetic beads |
| CN112557649A (en) * | 2019-09-25 | 2021-03-26 | 深圳光彩生命工程技术有限公司 | Method for detecting exosomes |
| CN114469996A (en) * | 2021-12-23 | 2022-05-13 | 中国医学科学院医学生物学研究所 | MiR-135b-5 p-containing exosome and application thereof in rotavirus infection resistance |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107271655A (en) * | 2017-05-18 | 2017-10-20 | 成都中医药大学附属医院 | A kind of kit and method for detecting urine excretion body load miRNAs |
| CN112557649A (en) * | 2019-09-25 | 2021-03-26 | 深圳光彩生命工程技术有限公司 | Method for detecting exosomes |
| CN110964694A (en) * | 2019-11-11 | 2020-04-07 | 浙江卫未生物医药科技有限公司 | Method for extracting exosome based on density gradient centrifugation and ultracentrifugation |
| CN112126643A (en) * | 2020-09-11 | 2020-12-25 | 上海长征医院 | Method for separating ecDNA (deoxyribose nucleic acid) in exosome based on magnetic beads |
| CN112126643B (en) * | 2020-09-11 | 2022-04-15 | 上海长征医院 | Method for separating ecDNA (deoxyribose nucleic acid) in exosome based on magnetic beads |
| CN114469996A (en) * | 2021-12-23 | 2022-05-13 | 中国医学科学院医学生物学研究所 | MiR-135b-5 p-containing exosome and application thereof in rotavirus infection resistance |
| CN114469996B (en) * | 2021-12-23 | 2023-10-20 | 中国医学科学院医学生物学研究所 | Exosomes containing miR-135b-5p and application thereof in resisting rotavirus infection |
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