CN109266613A - A kind of excretion body method for separating and preparing - Google Patents
A kind of excretion body method for separating and preparing Download PDFInfo
- Publication number
- CN109266613A CN109266613A CN201811155640.4A CN201811155640A CN109266613A CN 109266613 A CN109266613 A CN 109266613A CN 201811155640 A CN201811155640 A CN 201811155640A CN 109266613 A CN109266613 A CN 109266613A
- Authority
- CN
- China
- Prior art keywords
- excretion body
- liquid
- centrifuged
- added
- pillar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of excretion body method for separating and preparing, belong to field of biotechnology.A kind of excretion body method for separating and preparing includes the following steps: 1) centrifugation removal cell impurities；2) centrifugation removal cell fragment and big protein impurities；3) supernatant is transferred in new centrifuge tube, precipitating is abandoned in centrifugation, stays supernatant；4) it filters supernatant and shifts；5) it is transferred in film adsorption column, is centrifuged, the liquid at pipe abandon bottom after mixing reagent XBP with sample；6) centrifugation removal remains in the liquid on film；7) reagent XWP, centrifugation is added, the liquid at pipe abandon bottom is changed to pillar in new centrifuge tube；8) reagent XE is added on pillar, centrifugation elutes excretion body, obtains the liquid containing excretion body.The RNA purity for the excretion body that the present invention extracts improves, and impurity is few, can lower starting and extract the biological sample volume of excretion body while improving the yield of circulation excretion body.
Description
Technical field
The present invention relates to a kind of excretion body method for separating and preparing, belong to field of biotechnology.
Background technique
Huppert's disease (MM) is the common hematological system tumor of mid-aged population, accounts about all malignant tumours
1%, the 10% of Malignancy.As China progresses into aging, disease incidence is in obvious ascendant trend in recent years.
To the gradually intensification that MM pathogenesis and biological behaviour understand, a variety of effective therapeutic agents enter clinical application, and MM is
From a kind of tumour that therapeutic response rate is low, becomes treatment means multiplicity, can treat, controllable disease.But current MM is still
A kind of malignant tumour that can not be cured, almost all of MM can recur and finally invalid to treatment.
MM main, most basic diagnosis and prognostic evaluation methods are all based on bone marrow needle biopsy at present, the inspection wound
Property is big, and patient's especially gerontal patient's compliance is poor, is unfavorable for the early detection of disease；Furthermore there are more lesions to be dispersed in for MM morbidity
The feature of distribution, single site bone marrow aspiration can not react the general characteristic of patient tumors cell, this also become MM fail to pinpoint a disease in diagnosis with
And accurate major reason is owed in state of an illness evaluation.Therefore simple and easy, low-wound early diagnosis, curative effect monitoring and prognosis are researched and developed
The biomarker of evaluation has MM clinic diagnosis important theory and realistic meaning.
Excretion body (exosome) is to participate in the film property vesica that the diameter of cell-tocell exchange is 30-150nm, is inside taken
The nucleic acid of band and the physiology and pathologic function of protein influence recipient cell.MiRNA is the endogenous that a kind of length is 18-22nt
Single-stranded non-coding RNA passes through post-transcriptional control gene expression.It has been defined that, miRNA molecule is except participation normal cell growth, hair
It educates, be proliferated, outside apoptosis, also playing an important role in the occurrence and development of tumour.Excretion body be widely present in serum, blood plasma,
In the body fluid such as marrow slurry, urine, research in recent years finds in body fluid that excretion body is its main existence form there are miRNA molecule.
Since serum, plasma sample acquisition are easy and repeat to obtain, be conducive to the clinical tracing study to disease, and miRNA has
The advantage for having stability good, not degradable, therefore recycling miRNA is ideal Noninvasive biomarker, is had important
Application value and prospect.However excretion body separation method respectively has advantage and disadvantage at present, there is no unified, efficient isolation and purification method,
Seriously affect the consistency of extraction efficiency and later period miRNA analysis result.
Currently, the method for ultracentrifugation and film adsorption column can effectively obtain excretion body, but every kind of method has
Advantage and disadvantage.Time-consuming and requires larger amount of initial serum sample volume for ultracentrifugal method, since ultracentrifugation step is numerous
It is trivial, more excretion body is lost in separation process, therefore influence the yield of final exosome.Film adsorption column does not have sample volume
There is biggish requirement, extraction process is simple time-consuming short, and point of excretion body or excretion nucleic acid in vivo molecule can be completed in a hour
It is relatively convenient method for large batch of sample extraction excretion body from process.But its dedoping step is simpler, have compared with
More impurity residuals, therefore subsequent experimental will be polluted, such as remaining cell protein, nucleic acid ingredient.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of improved excretion body method for separating and preparing.
A kind of excretion body method for separating and preparing, includes the following steps:
1) cell impurities in centrifugation removal biological sample；
2) centrifugation removal cell fragment and big protein impurities；
3) supernatant is transferred in new 1.5ml centrifuge tube, 4 DEG C, 12000g is centrifuged 45min, abandons precipitating, stays supernatant；
4) supernatant is filtered with 0.2 μm of filter and be transferred in new 1.5ml centrifuge tube；
5) film adsorption column exoEasy Midi is transferred to after mixing reagent XBP and sample according to the ratio of volume ratio 1: 1
In Spin Columns, 500g is centrifuged 1min, the liquid at pipe abandon bottom；
6) liquid that 1min removal remains on exoEasy Midi Spin Columns film is centrifuged under conditions of 3500g
Body；
7) it is centrifuged under the conditions of addition 3.5ml reagent XWP, 3500g into pillar exoEasy Midi Spin Columns again
5min, the liquid at pipe abandon bottom are changed to pillar in new centrifuge tube；
8) the reagent XE of 400ul (QIAGEN No.160011905) is added to pillar exoEasy Midi Spin
Columns is upper to elute excretion body at 3500g, the centrifugal condition of 5min, obtains the liquid containing excretion body.
Cell impurities in the step 1) centrifugation removal biological sample refer to peripheral blood or marrow slurry at 4 DEG C, 2500g
Under conditions of be centrifuged 10min, remove red blood cell, the influence of the cell impurities such as leucocyte, reject precipitating retains supernatant, supernatant is turned
It moves on in new 1.5ml EP pipe.
Step 2) the centrifugation removes cell fragment and big protein impurities refer to 500ul supernatant at 4 DEG C, 2000g's
Under the conditions of be centrifuged 30min, remove cell fragment and big protein impurities, abandon precipitating and stay supernatant.
The reagent XBP, reagent XWP, film adsorption column exoEasy Midi Spin Columns derive from kit
QIAGEN exoRNeasy Serum/Plasma Midi Kits cat nos:77044.
The biological sample is that blood or marrow are starched.
The biological sample is the biological sample of mammal.
The mammal is behaved.
The method also includes RNA extraction steps.
The RNA extraction step is as follows:
(1) 700 μ l QIAzol are added in the re-suspension liquid containing excretion body；
(2) after standing 5min at EP pipe being vortexed 5 seconds, 15 DEG C -25 DEG C, 90 μ l chloroforms is added and firmly rock 15s, room up and down
Temperature stands 2-3min；
(3) 12000g, 4 DEG C of centrifugation 15min；
(4) upper strata aqueous phase is transferred in new EP pipe, avoids being drawn onto intermediate organic layer；
(5) 100% long-pending ethyl alcohol of upper strata aqueous phase diploid is added, and (e.g., 800 μ l should be added in the upper strata aqueous phase of 400 μ l
100% ethyl alcohol), it is mixed by upper and lower pressure-vaccum, the liquid for drawing 700 μ l is added to pillar RNeasy MinElute Spin
It on Column, closes the lid, 8000g is centrifuged 15s, discards the liquid of tube bottom；
(6) step (5) are repeated with remaining liquid, until all liquid is shifted；
(7) the buffer RWT of 700 μ l is added on pillar, is closed the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
(8) the buffer RPE of 500 μ l is added on pillar, is closed the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
(9) the buffer RPE of 500 μ l is added on pillar, is closed the lid, 8000g is centrifuged 2min, the liquid at pipe abandon bottom
Body；
(10) RNeasy MinElute Spin Column is transferred in new 2ml collecting pipe, opens lid, with
The revolving speed of 14000g is centrifuged 5min, removes liquid remaining on pillar；
(11) RNeasy MinElute Spin Column is transferred in new 1.5ml centrifuge tube, by 14 μ l without RNA
The water of enzyme is added to the center membrane of pillar, is stored at room temperature 2min, and 14000g is centrifuged 2min, and the liquid at collecting pipe bottom obtains excretion
Internal RNA, and be stored in -80 DEG C of refrigerators.
The QIAzol, RNeasy MinElute Spin Column, buffer RWT, buffer RPE are from examination
Agent box QIAGEN exoRNeasy Serum/PlasmaMidi Kits cat No:77044.
Further, excretion body method for separating and preparing of the present invention is used for the detection of Huppert's disease.
Detection of the present invention refers to early diagnosis and/or prognostic evaluation.
This method improves the standard operation of exoRNeasy Serum/Plasma Midi Kit, for example increases removal
The process of impurity, so that it is higher to extract obtained excretion body purity.
Supercentrifugation is combined with two methods of film absorption, is excellent by this research and utilization clinical patients periphery blood specimen
The advantages of a set of new exosome isolation and purification method is established in change, and this method combines supercentrifugation and film combined techniques, and have
The defect for overcoming the two of effect.After present invention optimization, the excretion body extracted, through flow cytometry, western
The identification such as blot detection and granularmetric analysis, it was demonstrated that successfully obtain excretion body molecule in blood circulation.The excretion body that the present invention extracts
It is improved compared with 1 purity of comparative example, impurity is reduced, and can be lowered initial serum using volume, the yield of circulation excretion body be improved, from electricity
The results show that in the case where reducing serum volume, the method for film adsorption column obtains more mirror compared with the method for differential centrifugation
Excretion body.
The present invention will be further described with reference to the accompanying drawings and detailed description, not limitation of the invention.It is all
Any this field equivalent replacement carried out according to present disclosure, all belongs to the scope of protection of the present invention.
Detailed description of the invention
Fig. 1 is the quality inspection result for the excretion body RNA that excretion body separating and extracting process of the present invention obtains
Fig. 2 is the protein immunoblot test result for the excretion body that the method for the present invention is extracted
Fig. 3 is photo of the excretion body of the method for the present invention acquisition under Electronic Speculum
Fig. 4 is the quality inspection result for the RNA that 1 differential centrifugation of comparative example extracts excretion body
Fig. 5 is the detected by Western blot result that 1 differential centrifugation of comparative example extracts excretion body
Fig. 6 is that 1 differential centrifugation of comparative example extracts photo of the excretion body under Electronic Speculum
Fig. 7 is the quality inspection result for the RNA that 2 film absorption method of comparative example extracts excretion body
Fig. 8 A and Fig. 8 B are the detected by Western blot result that 2 film absorption method of comparative example extracts excretion body
Fig. 9 is that 2 film absorption method of comparative example extracts photo of the excretion body under Electronic Speculum
Specific embodiment
A kind of embodiment 1: excretion body method for separating and preparing
One, material
Peripheral blood: the discarded sample after patient clinical inspection
Marrow slurry: the discarded sample after patient clinical inspection
Reagent XBP, reagent XWP, film adsorption column exoEasy Midi Spin Columns, pillar Spin Column derives from kit QIAGEN exoRNeasy Serum/PlasmaMidi Kits cat
No:77044.
Reagent XE:QIAGEN Lot No.160011905
Excretion body RNA extracts kit: exoRNeasy Serum/Plasma Midi Kit, German Qiagen company,
cat.No.77044
Two, methods
1. centrifugation removal cell impurities: by peripheral blood or marrow slurry at 4 DEG C, 10min is centrifuged under conditions of 2500g, is removed
The influence of the cell impurities such as red blood cell, leucocyte, reject precipitating retain supernatant, 500 μ l supernatants are transferred to new 1.5ml EP
Guan Zhong.
2. centrifugation removal cell fragment and big protein impurities: by 500 μ l supernatants at 4 DEG C, being centrifuged under conditions of 2000g
30min removes cell fragment and big protein impurities, abandons precipitating and stays supernatant.
3. supernatant is transferred in new 1.5ml centrifuge tube, 4 DEG C, 12,000g centrifugation 45min abandon precipitating, stay supernatant；
4. with 0.2 μm of filter filtering supernatant and being transferred in new EP centrifuge tube；
5. being transferred to film adsorption column exoEasy Midi after reagent XBP and sample are mixed according to the ratio of volume ratio 1: 1
In Spin Columns, 500g is centrifuged 1min, the liquid at pipe abandon bottom；
6. being centrifuged the liquid that 1min removal remains on exoEasy Midi Spin Columns film under conditions of 3500g
Body；
7. being centrifuged under the conditions of 3.5ml reagent XWP, 3500g is added into pillar exoEasy Midi Spin Columns again
5min, the liquid at pipe abandon bottom are changed to pillar in new centrifuge tube；
8. the reagent XE (QIAGEN Lot No.160011905) of 400 μ l is added to pillar (exoEasy Midi Spin
Columns excretion body is eluted at 3500g, the centrifugal condition of 5min on), obtains the liquid containing excretion body.
Three, results
Obtain the re-suspension liquid containing excretion body of derived from peripheral blood.
Obtain the re-suspension liquid containing excretion body in marrow slurry source.
Embodiment 2: excretion body total serum IgE is extracted
One, material
Embodiment 1 obtains the liquid sample containing excretion body
Excretion body RNA extracts kit: exoRNeasy Serum/Plasma Midi Kit, German Qiagen company,
cat.No.77044
Two, methods
1. 700 μ l QIAzol are added in the re-suspension liquid obtained above containing excretion body
2. EP pipe is vortexed 5 seconds, after (15 DEG C -25 DEG C) standing 5min of room temperature, 90 μ l chloroforms is added and firmly rock up and down
15s is stored at room temperature 2-3min；
3.12000g 4 DEG C of centrifugation 15min；
4. upper strata aqueous phase is transferred in new EP pipe, avoid being drawn onto intermediate organic layer；
5. 100% long-pending ethyl alcohol of upper strata aqueous phase diploid is added, and (e.g., 800 μ l 100% should be added in the upper strata aqueous phase of 400 μ l
Ethyl alcohol), it is mixed by upper and lower pressure-vaccum, the liquid for drawing 700 μ l is added to pillar (RNeasy MinElute Spin Column)
On, it closes the lid, 8000g is centrifuged 15s, discards the liquid of tube bottom；
6. step 5 is repeated with remaining liquid, until all liquid is shifted；
7. the buffer RWT of 700 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
8. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
9. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 2min, the liquid at pipe abandon bottom；
10. RNeasy MinElute Spin Column is transferred in new 2ml collecting pipe, lid is opened, with
The revolving speed of 14000g is centrifuged 5min, removes liquid remaining on pillar；
11. RNeasy MinElute Spin Column is transferred in new 1.5ml centrifuge tube, by 14 μ l without RNA enzyme
Water be added to the center membrane of pillar, be stored at room temperature 2min, 14000g is centrifuged 2min, and the liquid at collecting pipe bottom obtains excretion body
Interior RNA, and be stored in -80 DEG C of refrigerators.
Three, results
Excretion body total serum IgE is obtained, and is stored in -80 DEG C of refrigerators (sample number sample7:Q-5).
RNA quality testing in 3. excretion body of embodiment and excretion body
One, material
The excretion body sample (sample number Exo-6) that embodiment 1 obtains
RNA (sample number sample7:Q-5) in the excretion body that embodiment 2 obtains
CD63, TSG101, Calnexin (are purchased from Abcam company)
Marker: it is purchased from Thermo Scientific (#26617)
Pvdf membrane: it is purchased from Immobilon-P (Cat No.IPVH00010)
Primary antibody dilution: it is purchased from Beyotime (P0023A)
HELA: it is provided by Chinese Academy of Medical Sciences's basic research
TBST: being that 0.1% Tween-20 is added to configure by 10 × TBS
10 × TBS buffer:
Tris Base 24.2g
NaCl 80g
ddH2O 1000ml
TBST buffer:
10×TBS 100ml
ddH2O 900ml
Tween-20 1ml
Two, methods-
(1) RNA quality inspection
Hua Da gene studies chief minister is entrusted to carry out RNA quality inspection with 2100 biological analyser of Agilent, it is believed that in 25-
There is peak to show to have extracted exosome RNA between 200nt.
(2) detected by Western blot detection excretion body labelled protein (Western Blot)
1. cracking 30min on ice after being mixed with 20 μ l RIPA and 20 μ l excretion body samples, measuring excretion body with BCA
Protein concentration；
2. 5min is boiled after being mixed with 5 × SDS sample-loading buffer and excretion body at 95 DEG C, wink from, loading volume is 20 μ l,
Marker loading is 10 1 × SDS of μ l marker+10 μ l；
3. voltage is adjusted to 100v after the band for running out of marker with 70v voltage, total time is about 120min；
4. it is simultaneously dipped into methanol about 5min by the pvdf membrane for cutting 8cm × 5cm, with 250mA transferring film 2h, and with 5%
Skim milk (5g skimmed milk power+100ml TBST) room temperature close 1h；
5.CD63, TSG101, Calnexin antibody (being purchased from Abcam company) are diluted through primary antibody dilution, and CD63 is pressed
According to 1: 500 dilution proportion, TSG101 and Calnexin according to 1: 1000 dilution proportion.Film is put into primary antibody dilution, 4
DEG C it is incubated overnight primary antibody；
6. second day, the pvdf membrane after being incubated for primary antibody with TBST washed 3 times at room temperature, every time 10min, with
Afterwards, pvdf membrane is put into secondary antibody (secondary antibody is the antibody of anti-rabbit, is purchased from CST company), is incubated for 1h at room temperature, uses TBST
The film of secondary antibody will be incubated at room temperature washing 3 times, it is every all over 10min, it can develop.
(3) form of transmission electron microscope observation excretion body
Fig. 3 is photo (amplification factor 80kv) of the excretion body of the method for the present invention acquisition under Electronic Speculum.According to Electronic Speculum
As a result, it can be seen that under 0.5ml serum volume and 80kv amplification factor, the method for the present invention can obtain enough for Electronic Speculum
The excretion body of shooting.Excretion volume morphing is typical discoid, and diameter is in 100nm or so.
Three, results
(1) RNA quality inspection
As shown in Figure 1, excretion body method for separating and preparing of the invention can obtain relatively pure excretion body.The present invention
Method uses the serum of 500 μ l, can extract to obtain excretion body and can obtain wherein RNA molecule.
(2) detected by Western blot detects excretion body labelled protein
Western Blot result is as shown in Fig. 2, wherein Exo-6 is extraction excretion body progress WB verifying after present invention improvement
As a result, wherein Calnexin Protein Detection is feminine gender, illustrate that cell impurities removal is clean, and detect the mark egg of excretion body
White (CD63, TSG101).The result illustrates that this method can obtain the excretion body that impurity is few, with high purity.(Exo-4 and Exo-5 are film
Adsorption column method extract excretion body (comparative example 2) verifying as a result, showing Calnexin impurity protein bands)
Comparative example 1: differential centrifugation extracts RNA in excretion body and excretion body
One, material
Human peripheral: with embodiment 1
Ultracentrifugation pipe: Beckman company (344058) are purchased from
CD63, TSG101, Calnexin (are purchased from Abcam company)
Marker: with embodiment 3
Pvdf membrane: with embodiment 3
Primary antibody dilution: with embodiment 3
TBST: with embodiment 3
Two, methods
(1) excretion body is separated
1. 2500g is centrifuged 10min, removes red blood cell, the cells such as leucocyte by peripheral blood (whole blood) or marrow slurry at 4 DEG C
Impurity effect retains supernatant；
2. 4 DEG C, 2000g is centrifuged 30min, and removal cell is broken by 1ml supernatant and PBS according to the dilution proportion of volume ratio 1: 1
Piece and big albumen abandon precipitating；
3. supernatant is transferred in high speed centrifugation pipe, the PBS of addition and supernatant equivalent in high speed centrifugation pipe, 4 DEG C, 12,
000g is centrifuged 45min, retains supernatant, abandons precipitating；
4. with 0.2 μm of filter filtering supernatant and being transferred in ultracentrifugation pipe, liquid is mended to away from nozzle < 2cm with PBS；
5.4 DEG C, 130,000g centrifugation 2h abandon supernatant and stay precipitating；
6. PBS is added to be resuspended after excretion body, 4 DEG C, 130,000g, 2h centrifugation abandons supernatant, stays precipitating；
7. being stored in -80 DEG C after being hanged excretion weight with 100 μ l PBS.
(2) RNA in excretion body is extracted
1. 700 μ l QIAzol are added in the re-suspension liquid obtained above containing excretion body, centrifuge tube is vortexed 5 seconds, room
After (15 DEG C -25 DEG C) standing 5min of temperature, 90 μ l chloroforms are added and firmly rock 15s up and down, are stored at room temperature 2-3min
2.12000g 4 DEG C of centrifugation 15min；
3. upper strata aqueous phase is transferred in new EP pipe, avoid being drawn onto intermediate organic layer；
4. upper strata aqueous phase diploid product dehydrated alcohol is added, and (e.g., the 800 anhydrous second of μ l should be added in the upper strata aqueous phase of 400 μ l
Alcohol), it is mixed by upper and lower pressure-vaccum；
5. the liquid for drawing 700 μ l is added on pillar (RNeasy MinElute Spin Column), close the lid,
8000g is centrifuged 15s, discards tube bottom liquid；
6. step 5 is repeated with remaining liquid, until all liquid is shifted；
7. the buffer RWT of 700 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, reject tube bottom liquid；
8. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, reject tube bottom liquid；
9. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 2min, reject tube bottom liquid
10. RNeasy MinElute Spin Column is transferred in new 2ml collecting pipe, lid is opened, with
The revolving speed of 14000g is centrifuged 5min, removes liquid remaining on pillar
11. RNeasy MinElute Spin Column is transferred in new 1.5ml centrifuge tube, by 14 μ l without RNA enzyme
Water be added to the center membrane of pillar, be stored at room temperature 2min, 14000g is centrifuged 2min, and the liquid at collecting pipe bottom is stored in -80 DEG C
In refrigerator.
(3) RNA quality inspection in excretion body and excretion body
1.RNA quality inspection
Hua Da gene studies chief minister is entrusted to carry out RNA quality inspection with 2100 biological analyser of Agilent, it is believed that in 25-
There is peak to show to have extracted exosome RNA between 200nt.Sample number is sample10:E-1.
2. detected by Western blot detects excretion body labelled protein (Western Blot)
(1) after being mixed with 20 μ l RIPA and 20 μ l excretion bodies (sample number into spectrum Exo-1), 30min is cracked on ice, is used
BCA measures the protein concentration of excretion body；
(2) boil 5min after being mixed with 5 × SDS sample-loading buffer and excretion body at 95 DEG C, wink from, loading volume is 20 μ l,
Marker loading is 10 1 × SDS of μ l marker+10 μ l；
(3) voltage is adjusted to 100v after the band for running out of marker with 70v voltage, total time is about 120min；
(4) it cuts the pvdf membrane of 8cm × 5cm and it is dipped into methanol about 5min, with 250mA transferring film 2h, and with 5%
Skim milk (5g skimmed milk power+100mlTBST) room temperature close 1h；
(5) CD63, TSG101, Calnexin antibody (being purchased from Abcam company) use primary antibody diluted, and CD63 is according to 1:
500 dilution proportion, TSG101 and Calnexin according to 1: 1000 dilution proportion.Above-mentioned pvdf membrane is put into primary antibody dilution
In, 4 DEG C of overnight incubations；
(6) second days, pvdf membrane is washed at room temperature 3 times with TBST, it is every then to put pvdf membrane all over 10min
Into secondary antibody (with embodiment 3), it is incubated for 1h at room temperature, later TBST room temperature washing 3 times, it is every all over 10min, Ji Kexian
Shadow.
(3) form of transmission electron microscope observation excretion body
Excretion body is dyed using phosphotungstic acid.
It is observed using Hitachi HT7700 projection electron Electronic Speculum.
Three, results
1.RNA quality inspection
Fig. 4 is the quality inspection result for the RNA that 1 differential centrifugation of comparative example extracts excretion body.Differential surpasses to be lacked from what method obtained
The RNA that the excretion body of amount extracts without the peak RNA, that is, there is no the RNA in excretion body after the detection of Agilent 2100.The result
Illustrating the serum using 1ml, surpasses through differential from a large amount of excretion body has been lost in method extraction process, excretion body yield is lower,
It is not able to satisfy the extraction of subsequent excretion body RNA.
2. detected by Western blot detects
Fig. 5 is that differential centrifugation extracts the detected by Western blot of excretion body as a result, Hela is positive control, sample number
For Exo-1.WB figure shows that differential surpasses and do not have impurity Calgexin in the excretion body sample obtained from method, and the excretion body of acquisition is miscellaneous
Matter is less, and purity is higher.
3. the form of transmission electron microscope observation excretion body
Fig. 6 is photo (amplification factor 80kv) of the excretion body of this comparative example acquisition under Electronic Speculum.According to the knot of Electronic Speculum
Fruit, it can be seen that under 1ml serum volume and 80kv amplification factor, ultracentrifugation extracts the method (comparative example 1) of excretion body simultaneously
Enough excretion bodies for Electronic Speculum shooting are not obtained.So ultracentrifugal method had lost compared with the method for film adsorption column it is more
Excretion body.
Comparative example 2: film adsorption column method extracts RNA in excretion body and excretion body
One, material
Human peripheral: with embodiment 1
Excretion body RNA extracts kit: exoRNeasy Serum/Plasma Midi Kit, German Qiagen company,
cat.No.77044
CD63, TSG101, Calnexin (are purchased from Abcam company)
Marker: with embodiment 3
Pvdf membrane: with embodiment 3
Primary antibody dilution: with embodiment 3
TBST: with embodiment 3
Two, methods
(1) excretion body is separated
1. 2500g is centrifuged 10min, removes red blood cell, the cells such as leucocyte by peripheral blood (whole blood) or marrow slurry at 4 DEG C
Impurity effect retains supernatant, 500 μ l supernatants is transferred in new centrifuge tube；
2. 500 μ l supernatants are filtered with 0.45 μm of filter and are transferred in new EP pipe；
2.16000g, 4 DEG C of centrifugation 10min abandon precipitating and retain supernatant；
3. being added in pillar after reagent XBP and sample are mixed according to the ratio of volume ratio 1: 1,500g is centrifuged 1min,
The liquid at pipe abandon bottom.(kit of used film adsorption column is QIAGEN exoRNeasy Serum/Plasma Midi Kits
Cat No:77044)
6.3500g, 1min removal remain in the liquid on film
7. 3.5ml reagent XWP is added into pillar (exoEasy Midi Spin Columns) again, 3500g centrifugation
5min, pipe abandon bottom liquid, pillar is changed in new centrifuge tube.
Excretion body is eluted at 3500g, the centrifugal condition of 5min 8. the reagent XE of 400 μ l is added on pillar, is obtained
The liquid sample containing excretion body.
(2) RNA in excretion body is extracted
1. 700 μ l QIAzol are added in the re-suspension liquid obtained above containing excretion body, centrifuge tube is vortexed 5 seconds, room
After (15 DEG C one 25 DEG C) standing 5min of temperature, 90 μ l chloroforms are added and firmly rock 15s up and down, are stored at room temperature 2-3min；
2.12000g 4 DEG C of centrifugation 15min；
3. upper strata aqueous phase is transferred in new EP pipe, avoid being drawn onto intermediate organic layer；
4. upper strata aqueous phase diploid product dehydrated alcohol is added, and (e.g., the 800 anhydrous second of μ l should be added in the upper strata aqueous phase of 400 μ l
Alcohol), it is mixed by upper and lower pressure-vaccum, the liquid for drawing 700 μ l is added on pillar (RNeasy MinElute spin colum), is covered
Upper cover, 8000g are centrifuged 15s, discard tube bottom liquid；
5. step 4 is repeated with remaining liquid, until all liquid is shifted；
6. the buffer RWT of 700 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, reject tube bottom liquid；
7. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 15s, reject tube bottom liquid；
8. the buffer RPE of 500 μ l is added on pillar, close the lid, 8000g is centrifuged 2min, reject tube bottom liquid
9. RNeasy MinElute Spin Column is transferred in new 2ml collecting pipe, lid is opened, with
The revolving speed of 14000g is centrifuged 5min, removes liquid remaining on pillar
10. RNeasy MinElute Spin Column is transferred in new 1.5ml centrifuge tube, by 14 μ l without RNA enzyme
Water be added to the center membrane of pillar, be stored at room temperature 2min, 14000g is centrifuged 2min, the liquid at collecting pipe bottom, and is stored in -80
In DEG C refrigerator.
(3) RNA quality inspection in excretion body and excretion body
1.RNA quality inspection
Hua Da gene studies chief minister is entrusted to carry out RNA quality inspection with 2100 biological analyser of Agilent, it is believed that in 25-
There is peak to show to have extracted RNA between 200nt.Sample number is sample4:Q-1.
2. detected by Western blot detects excretion body labelled protein (Western Blot)
(1) it after being mixed with 20 μ l RIPA and 20 μ l excretion bodies (sample number is Exo-2 and Exo-3), cracks on ice
30min measures the protein concentration of excretion body with BCA；
(2) boil 5min after being mixed with 5 × SDS sample-loading buffer and excretion body at 95 DEG C, wink from, loading volume is 20 μ l,
Marker loading is 10 1 × SDS of μ l marker+10 μ l；
(3) voltage is adjusted to 100v after the band for running out of marker with 70v voltage, total time is about 120min；
(4) it cuts the pvdf membrane of 8cm × 5cm and it is dipped into methanol about 5min, with 250mA transferring film 2h, and with 5%
Skim milk (5g skimmed milk power+100mlTBST) room temperature close 1h；
(5) CD63, TSG101, Calnexin (antibody of Abcam) primary antibody diluted, CD63 is according to 1: 500
Dilution proportion, TSG101 and Calnexin according to 1: 1000 dilution proportion.Pvdf membrane is put into primary antibody dilution, 4 DEG C incubate
It educates overnight；
(6) second days, the film for being incubated for primary antibody is washed 3 times in room temperature with TBST, it is every that film is then put into two all over 10min
(with embodiment 3) in anti-, it is incubated for 1h at room temperature, is washed the film for being incubated for secondary antibody 3 times, every time in room temperature with TBST
10min can develop.
3. the form of transmission electron microscope observation excretion body
Phosphotungstic acid carries out dyeing processing to excretion body
It is observed using Hitachi's HT7700 projection electron microscope.
Three, results
1.RNA quality inspection
Fig. 7 is the quality inspection result for the RNA that film adsorption column method extracts excretion body.Using 0.5ml serum, film adsorption column method is obtained
Excretion body extract RNA through Agilent 2100 detection after have the peak RNA, RNA can be obtained.
2. detected by Western blot detects
Fig. 8 A and Fig. 8 B are that film adsorption column method extracts the detected by Western blot of excretion body as a result, Hela is positive control,
Sample is Exo-2 and Exo-3.WB figure is shown, in the excretion body sample that film adsorption column method obtains, the impurity band ratio of Calnexin
It is more apparent, so there is the doping of protein impurities in the excretion body that film adsorption column extracts.The excretion body impurity of acquisition is more, purity compared with
It is low.
3. the form of transmission electron microscope observation excretion body
Fig. 9 is photo (amplification factor 80kv) of the excretion body of this comparative example acquisition under Electronic Speculum.According to the knot of Electronic Speculum
Fruit, it can be seen that under 0.5ml initial serum volume and 80kv amplification factor, film adsorption column extracts the method for excretion body in Electronic Speculum
It can be seen that a large amount of excretion body in figure.
Claims (10)
1. a kind of excretion body method for separating and preparing, which comprises the steps of:
1) cell impurities in centrifugation removal biological sample；
2) centrifugation removal cell fragment and big protein impurities；
3) supernatant is transferred in new 1.5ml centrifuge tube, 4 DEG C, 12000g is centrifuged 45min, abandons precipitating, stays supernatant；
4) supernatant is filtered with 0.2 μm of filter and be transferred in new 1.5ml centrifuge tube；
5) film adsorption column exoEasy Midi Spin is transferred to after mixing reagent XBP and sample according to the ratio of volume ratio 1: 1
In Columns, 500g is centrifuged 1min, the liquid at pipe abandon bottom；
6) liquid that 1min removal remains on exoEasy Midi Spin Columns film is centrifuged under conditions of 3500g；
7) it is centrifuged under the conditions of addition 3.5ml reagent XWP, 3500g into pillar exoEasy Midi Spin Columns again
5min, the liquid at pipe abandon bottom are changed to pillar in new centrifuge tube；
8) the reagent XE (QIAGEN No.160011905) of 400 μ l is added on exoEasy Midi Spin Columns
Excretion body is eluted under the centrifugal condition of 3500g, 5min, obtains the liquid containing excretion body.
2. a kind of excretion body method for separating and preparing according to claim 1, it is characterised in that: step 1) the centrifugation removal
Cell impurities in biological sample refer to peripheral blood or marrow slurry at 4 DEG C, and 10min is centrifuged under conditions of 2500g, remove red thin
Born of the same parents, the influence of the cell impurities such as leucocyte, reject precipitating retain supernatant, supernatant are transferred in new 1.5ml EP pipe.
3. a kind of excretion body method for separating and preparing according to claim 1, it is characterised in that: step 2) the centrifugation removal
Cell fragment and big protein impurities refer to supernatant at 4 DEG C, are centrifuged 30min under conditions of 2000g, removal cell fragment and big
Protein impurities, abandon precipitating stay supernatant.
4. a kind of excretion body method for separating and preparing according to claim 1, it is characterised in that: the reagent XBP, reagent
XWP, film adsorption column exoEasy Midi Spin Columns derive from kit QIAGEN exoRNeasy Serum/
Plasma Midi Kits cat No:77044.
5. according to claim 1 to excretion body method for separating and preparing described in any one of 4, it is characterised in that: the biology
Sample is that blood or marrow are starched.
6. excretion body method for separating and preparing according to claim 5, it is characterised in that: the biological sample is mammal
Biological sample.
7. excretion body method for separating and preparing according to claim 5, it is characterised in that: the mammal is behaved.
8. excretion body method for separating and preparing according to claim 7, it is characterised in that: further include RNA extraction step.
9. excretion body method for separating and preparing according to claim 8, which is characterized in that the RNA extraction step is as follows:
(1) 700 μ l QIAzol are added in the re-suspension liquid containing excretion body；
(2) after standing 5min at EP pipe being vortexed 5 seconds, 15 DEG C -25 DEG C, 90 μ l chloroforms is added and firmly rock 15s up and down, room temperature is quiet
Set 2-3min；
(3) 12000g, 4 DEG C of centrifugation 15min；
(4) upper strata aqueous phase is transferred in new EP pipe, avoids being drawn onto intermediate organic layer；
(5) 100% long-pending ethyl alcohol of upper strata aqueous phase diploid is added, and (e.g., 800 μ l, 100% second should be added in the upper strata aqueous phase of 400 μ l
Alcohol), it is mixed by upper and lower pressure-vaccum, the liquid for drawing 700 μ l is added on pillar RNeasy MinElute Spin Column, is covered
Upper cover, 8000g are centrifuged 15s, discard the liquid of tube bottom；
(6) step (5) are repeated with remaining liquid, until all liquid is shifted；
(7) the buffer RWT of 700 μ l is added on pillar, is closed the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
(8) the buffer RPE of 500 μ l is added on pillar, is closed the lid, 8000g is centrifuged 15s, the liquid at pipe abandon bottom；
(9) the buffer RPE of 500 μ l is added on pillar, is closed the lid, 8000g is centrifuged 2min, the liquid at pipe abandon bottom；
(10) RNeasy MinElute Spin Column is transferred in new 2ml collecting pipe, lid is opened, with 14000g
Revolving speed be centrifuged 5min, remove remaining liquid on pillar；
(11) RNeasy MinElute Spin Column is transferred in new 1.5ml centrifuge tube, by 14 μ l without RNA enzyme
Water is added to the center membrane of pillar, is stored at room temperature 2min, and 14000g is centrifuged 2min, and the liquid at collecting pipe bottom obtains in excretion body
RNA, and be stored in -80 DEG C of refrigerators.
10. excretion body method for separating and preparing according to claim 9, it is characterised in that: described QIAzol, RNeasy
MinElute Spin Column, buffer RWT, buffer RPE derive from kit QIAGEN exoRNeasy Serum/
Plasma Midi Kits cat No:77044.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811155640.4A CN109266613A (en) | 2018-09-29 | 2018-09-29 | A kind of excretion body method for separating and preparing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811155640.4A CN109266613A (en) | 2018-09-29 | 2018-09-29 | A kind of excretion body method for separating and preparing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109266613A true CN109266613A (en) | 2019-01-25 |
Family
ID=65195371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811155640.4A Pending CN109266613A (en) | 2018-09-29 | 2018-09-29 | A kind of excretion body method for separating and preparing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109266613A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551680A (en) * | 2019-08-12 | 2019-12-10 | 远辰生物科技(苏州)有限公司 | Method and system for extracting pleural effusion exosomes |
| CN111073846A (en) * | 2019-12-20 | 2020-04-28 | 江苏为真生物医药技术股份有限公司 | Method for separating extracellular vesicles from tissue specific sources and kit thereof |
| CN112147058A (en) * | 2019-06-26 | 2020-12-29 | 首都医科大学附属北京佑安医院 | Method for detecting hepatocyte-derived exosomes in plasma |
| EP4273229A4 (en) * | 2020-12-29 | 2024-12-04 | Union Korea Life Sciences Inc. | EXOSOME ISOLATION PROCESS WITH HIGH EFFICIENCY AND HIGH PURITY |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676065A (en) * | 2017-03-10 | 2017-05-17 | 四川大学 | Adipose tissue source exosome gel, preparation method and application |
| CN107201361A (en) * | 2017-05-11 | 2017-09-26 | 广州华银医学检验中心有限公司 | A kind of extracting method of excretion body total serum IgE |
| US20180187260A1 (en) * | 2015-06-17 | 2018-07-05 | The Translational Genomics Research Institute | Systems and methods for obtaining biological molecules from a sample |
-
2018
- 2018-09-29 CN CN201811155640.4A patent/CN109266613A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180187260A1 (en) * | 2015-06-17 | 2018-07-05 | The Translational Genomics Research Institute | Systems and methods for obtaining biological molecules from a sample |
| CN106676065A (en) * | 2017-03-10 | 2017-05-17 | 四川大学 | Adipose tissue source exosome gel, preparation method and application |
| CN107201361A (en) * | 2017-05-11 | 2017-09-26 | 广州华银医学检验中心有限公司 | A kind of extracting method of excretion body total serum IgE |
Non-Patent Citations (3)
| Title |
|---|
| QIAGEN: "exoEasy Maxi Kit Handbook", 《HTTPS://WWW.QIAGEN.COM/US/RESOURCES/DOWNLOAD.ASPX?ID=2F6FDA57-C00F-4425-9705-9F8795AAD12B&LANG=EN》 * |
| QIAGEN: "Purification of exosomal RNA from urine using the exoRNeasy Serum/Plasma Midi/Maxi Kit", 《HTTPS://WWW.QIAGEN.COM/US/PRODUCTS/DISCOVERY-AND-TRANSLATIONAL-RESEARCH/EXOSOMES-CTCS/EXOSOMES/EXORNEASY-MIDI-AND-MAXI-KITS/》 * |
| 江连洲: "《酶在大豆制品中的应用》", 3 August 2015, 中国轻工业出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112147058A (en) * | 2019-06-26 | 2020-12-29 | 首都医科大学附属北京佑安医院 | Method for detecting hepatocyte-derived exosomes in plasma |
| CN110551680A (en) * | 2019-08-12 | 2019-12-10 | 远辰生物科技(苏州)有限公司 | Method and system for extracting pleural effusion exosomes |
| CN111073846A (en) * | 2019-12-20 | 2020-04-28 | 江苏为真生物医药技术股份有限公司 | Method for separating extracellular vesicles from tissue specific sources and kit thereof |
| CN111073846B (en) * | 2019-12-20 | 2022-06-28 | 江苏为真生物医药技术股份有限公司 | Method for separating extracellular vesicles from tissue specific sources and kit thereof |
| EP4273229A4 (en) * | 2020-12-29 | 2024-12-04 | Union Korea Life Sciences Inc. | EXOSOME ISOLATION PROCESS WITH HIGH EFFICIENCY AND HIGH PURITY |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109266613A (en) | A kind of excretion body method for separating and preparing | |
| CN106591428B (en) | Detection and application of novel gastric cancer molecular marker hsa _ circ _0001017 | |
| CN107254519A (en) | The screening technique of triple negative breast cancer specificity circular rna | |
| CN111004850A (en) | Application of circRNAs molecules in preparation of liver cancer diagnosis kit and kit applying molecules | |
| CN110511902A (en) | Based on the separation of the extracellular vesica of exclusion chromatography and hyperfiltration technique and enrichment method | |
| CN102495146A (en) | Compound fingerprint atlas model used in early-stage gastric cancer diagnosis/early warning, and establishing method thereof | |
| CN107815495A (en) | A kind of blood plasma circular rna marker detection method related to non-small cell lung cancer | |
| CN107012145A (en) | A kind of long non-coding RNA and its application in diagnosis/treatment cholangiocarcinoma | |
| CN105861672A (en) | Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA | |
| CN118731362B (en) | Application of EF1G as a molecular marker in the preparation of products for diagnosing benign prostatic hyperplasia | |
| CN102707058A (en) | Tumor necrosis factor-alpha induced protein 8 L3 (TIPE3) immunohistochemistry detection kit for diagnosing lung cancer | |
| CN111961636B (en) | Exosome extraction reagent and application thereof | |
| CN120290726A (en) | A blood marker for diagnosing cervical cancer and its application | |
| CN119506365A (en) | A preparation method of anti-inflammatory compound tetramethyl betaine | |
| CN108342487B (en) | Esophageal cancer diagnosis specific expression map and detection analysis system based on serum exosomal lncRNAs | |
| CN111575385B (en) | Application of SB290157 in ovarian epithelial cancer diseases | |
| CN116732176A (en) | Application of gene translocation type renal cell carcinoma marker group | |
| CN117482923A (en) | Hollow glass microspheres densely packed with metal-organic frameworks and their preparation and application | |
| CN102286431A (en) | Monoclonal antibody for resisting Japanese encephalitis virus (JEV) and application thereof | |
| CN209485829U (en) | A device for enriching urine exosomes that can be used in hospitals and homes | |
| CN114231626A (en) | Application of miRNA (micro ribonucleic acid) combined marker in preparation of kit for diagnosing and detecting early liver cancer | |
| CN114774541B (en) | A plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and its application | |
| CN111303103A (en) | Preparation method and application of total coumarins of starburst canopy vine | |
| CN111638354A (en) | Immunofluorescence kit for detecting expression of peripheral blood circulating tumor cells E-Cadherin of pancreatic cancer patient | |
| CN112147336A (en) | A marker for detecting and evaluating liver regeneration and its use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190125 |