CN109439757A - Application of the blood plasma excretion body miR-455-3p as early liver cancer diagnosis marker - Google Patents
Application of the blood plasma excretion body miR-455-3p as early liver cancer diagnosis marker Download PDFInfo
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Abstract
The invention belongs to biotechnologys and medical domain, and in particular to a kind of application of the diagnosis marker of blood plasma excretion body miR-455-3p as liver cancer (early stage).
Description
Technical field
The invention belongs to biotechnologys and medical domain, and in particular to a kind of blood plasma excretion body miR-455-3p is as liver cancer
The application of the diagnosis marker of (early stage).
Background technique
Primary carcinoma of liver is one of most common malignant tumour in China, and disease incidence occupies the 4th in all malignant tumours
Position, and case fatality rate is in third position, wherein hepatocellular carcinoma (hepatocellular carcinoma, HCC) accounts for all primary
The 85%~90% of property liver cancer.HCC prognosis mala is mainly attributed to the pathogenic process in its concealment, therefore many patients are in head
As middle and advanced stage when examining, loses radical treatment chance.And AFP indicates its susceptibility as main serologic marker object
For 40-65%, specificity is 76-90%, there is nearly 1/3 liver cancer patient AFP negative.So AFP as early diagnosis index simultaneously
It is undesirable.Therefore the high HCC serologic marker object of sensibility is found, is not only played an important role in hepatocarcinoma early diagnosis,
It is significant simultaneously for the monitoring of early stage HCC root value criterion conditions of patients, recurrence prediction and prognosis evaluation etc..
Excretion body is released by cell secretion, is propagated in the body fluid such as blood, is the important medium of cell-cell communication.It is swollen
Tumor excretion body carry derive from tumour cell biological information (albumen, DNA and microRNA etc.), the concentration of content with
The invasive ability and tumor microenvironment of tumour cell are related, can be direct by the surface markers or internal component of analyzing excretion body
Obtain the essential information of cancer cell.Since excretion body has lipid film protection, content such as DNA, RNA and protein are not easy to be dropped
Solution is destroyed, so that no matter fresh or long-term preservation sample all can be used for analyzing.Importantly, excretion body can comform
(blood, urine etc.) is obtained in more body fluid, this makes excretion physical examination survey the great future in tumour diagnosis and treatment, becomes a kind of and more manages
" liquid biopsy " method thought.
Excretion body liver cancer diagnosis there are no specific relevant report, only document claims and traditional liver cancer marker
AFP is compared, in excretion body miRNA expression liver cancer early stage i.e. change and change level it is larger [Belov L1,
Matic KJ2,Hallal S2,Best OG2,3,Mulligan SP2,3,Christopherson RI2.Extensive
surface protein profiles of extracellular vesicles from cancer cells may
provide diagnostic signatures from blood samples.J Extracell Vesicles.2016Apr
15；5:25355], the diagnostic of early liver cancer screening is helped to improve with AFP combination.Just it is published in " Hepatology "
On studies have shown that microRNA let-7 in blood plasma and excretion body and chronic hepatitis C patient liver fibrosis progression have it is certain
Relationship [Circulating let-7levels in plasma and extracellular vesicles correlate
with hepatic fibr osis progression in chronic hepatitis C.Hepatology.2016May
26.doi:10.1002/hep. 28660].MiRNAs is found to take part in the generation of kinds cancer and development, miRNA expression with
Functional analysis proves that miRNA significantly affects the generation of tumour, and expression imbalance promotes the formation of malignant phenotype, becomes latent
Diagnosing tumor Index for diagnosis biomarker and oncotherapy potential target spot.The gene position of miR-455-3p.1
In No. 9 chromosomes, the loop-stem structure sequence generated from the transcription of mir-455 family encoding gene, mature sequence 5 '-
GCAGUCCAUGGGCAUAUACAC-3 ' is the microRNA comprising 21 nucleotide.It is previous research shows that miR-455-
3p takes part in such as biosynthesis of interference cholesterol of many benign pathological processes, inhibits myocardial hypertrophy after heart infarction, also assists in
The generation and development of the kinds of tumors such as kinds of tumors such as spongiocytoma, colon cancer, breast cancer, basal-cell carcinoma, such as miR-
455-3p is low expression in gastric cancer, may be played a significant role in control stomach organization angiogenic growth；MiR-455-3p can
Targeted-control PIK3R1 gene promotes clear cell carcinoma of kidney cell Proliferation, invasion and transfer；MiR-455-3p may pass through blocking
The migration and invasion of PI3K/AKT signal path inhibition colon cancer cell.But the relationship of itself and liver cancer had previously had not been reported.
The present invention analyzes the horizontal variation of the miR-455-3p in early liver cancer blood plasma excretion body, and provides blood plasma excretion
Application of the body miR-455-3p as liver cancer (early stage) diagnosis marker.
Summary of the invention
The purpose of the present invention is overcome the shortcomings of to provide a kind of blood plasma excretion body to diagnosing cancer of liver in the prior art
Application of the miRNA-455-3p as the diagnosis marker of liver cancer (early stage).
Technical solution: in order to achieve the goal above, the technical scheme adopted by the invention is as follows:
The present invention provides application of the blood plasma excretion body miRNA-455-3p as liver cancer (early stage) diagnosis marker, described
The sequence 5 '-GCAGUCCAUGGGCAUAUACAC-3 ' of miRNA-455-3p.
The gene of miR-455-3p.1 is located at No. 9 chromosomes, the stem generated from the transcription of mir-455 family encoding gene
Ring structure sequence, mature sequence 5 '-GCAGUCCAUGGGCAUAUACAC-3 ' are the small molecules comprising 21 nucleotide
RNA。
Excretion body is the double-deck film property vesicles of diameter about 30-150nm a kind of, and Various Tissues and cell can all secrete shape
At being present in the body fluid such as blood, cerebrospinal fluid, saliva and urine.They contain microRNA and albumen from host cell
The Multiple components such as matter are the important information interchange media of iuntercellular, while being also the reason of non-invasive disease diagnosis marker research
Think object.The invention will analyze expression of the miRNA-455-3p in blood plasma in early liver cancer patients blood plasma's excretion body, and visit
Beg for a possibility that it is as liver cancer (early stage) marker.
Extract the excretion body and excretion body miRNA in blood plasma, Fluorescent quantitative PCR (PCR) technology
Analyze expression and difference of the miRNA-455-3p in early liver cancer patient, patient with chronic HBV and healthy population blood plasma excretion body.
It is another object of the present invention to provide a kind of excretion body miRNA-455-3p for diagnosing liver cancer.
It is another object of the present invention to provide application of the excretion body miRNA-455-3p in terms of diagnosing liver cancer.
It is another object of the present invention to provide application of the excretion body miRNA-455-3p in terms of diagnosing early liver cancer.
It is another object of the present invention to provide excretion body miRNA-455-3p as tumor marker in diagnosis early stage
The application of liver cancer.
It is another object of the present invention to provide excretion body miRNA-455-3p to prepare early liver cancer diagnostic marker
Product in application.
The above-mentioned application of the present invention, specifically includes the following steps:
Application according to claim 5 or 6, which is characterized in that
1) preparation of plasma sample
EDTA anti-coagulants is added in heparin tube, after having acquired blood, heparin tube is slowly mixed by inversion, the whole blood of mixing
4 DEG C 1,000-2,000 × g is centrifuged 5-10min, and upper layer yellow translucent liquid is blood plasma to be collected, and pastes when blood plasma is sucked out
The page gradually inhale down, be sure not be sucked out cell component；The blood plasma being collected into can be directly used for subsequent experimental or dispense -80 DEG C
Refrigerator saves,
2) extraction of excretion body
By blood plasma room temperature, 2,000 × g is centrifuged 20min, removes residual cell and fragment；Supernatant is shifted to new centrifuge tube
In, it is careful not to be drawn onto bottom precipitation；Room temperature, 10,000 × g are centrifuged 20min, remove residual fragment；Supernatant is shifted with pipettor
Into new centrifuge tube, 500ul 1 × PBS and 300ulVEX Exosome Isolation is added in 1ml plasma sample
Mixed liquor is put in 2-8 DEG C of standing 30min after mixing vertically and is incubated for by Reagent solution, room temperature 10,000 × g centrifugation
5min removes supernatant, then room temperature 10, and 000 × g is centrifuged 30s, absorbs residual liquid with pipettor, blood plasma excretion body is present in pipe
In bottom precipitation,
3) extraction of excretion body miRNA
Excretion body miRNA, which is extracted, uses kit, and main operational steps are as follows: 1. taking excretion body, 1ml Trizol is added
Sufficiently homogenate, oscillator oscillation or pipettor suction are beaten and are mixed for several times, 5min are stored at room temperature, so that nucleic acid-protein compound is divided completely
From 2. 4 DEG C of 12,000rpm (13,400 × g) are centrifuged 5min, take supernatant, are transferred in the new centrifuge tube without RNase, 3.
200 μ l chloroforms are added, covers pipe lid, acutely vibrates 15sec, be placed at room temperature for 5min, 4. 4 DEG C of 12,000rpm (13,400 × g)
Be centrifuged 15min, sample can be divided into three layers: the organic phase of yellow, middle layer and colourless water phase, RNA is mainly in water phase, water phase
Volume be about the 50% of lysate MZ reagent used, water phase is transferred in new pipe, next step operation is carried out, 5. measures transfer
The volume of liquid is slowly added to transfer liquid 0.43 times of dehydrated alcohol of product, mixes, by obtained solution and precipitating be transferred to together to
In adsorption column miRspin, room temperature 12,000rpm (13,400 × g) is centrifuged 30sec, if once cannot be by complete soln and mixing
Object is added into adsorption column miRspin, is please transferred in two times, is discarded after centrifugation to adsorption column miRspin, retains efflux, 6.
The volume for measuring efflux, is slowly added to 0.75 times of effluent volume of dehydrated alcohol, mixes, by obtained solution and precipitating one
It rises and is transferred in adsorption column miRelute, room temperature 12,000rpm (13,400 × g) is centrifuged 30sec, 7. to adsorption column miRelute
500 μ l protein liquid removal MRD of middle addition are stored at room temperature 2min, room temperature 12, and 000rpm (13,400 × g) is centrifuged 30sec, abandon useless
8. 500 μ l rinsing liquid RW are added in liquid into adsorption column miRelute, be stored at room temperature 2min, room temperature 12,000rpm (13,400 ×
G) it is centrifuged 30sec, abandons waste liquid, 9. adsorption column miRelute, is 10. put into 2ml collecting pipe by repetitive operation step 7, room temperature 12,
000rpm (13,400 × g) is centrifuged 1min, removes residual liquid, adsorption column miRelute is transferred to a new RNase-
In 1.5 ml centrifuge tube of Free, adds 15 μ l RNase-FreeddH2O, be placed at room temperature for 2min, room temperature 12,000rpm (13,400
× g) centrifugation 2min,
4) cDNA is synthesized
Take 20ngRNA, based on ReverTra Ace qPCR RT Kit:FSQ-101, TOYOBO Reverse Transcriptase kit into
Row is added 5 × RT of 2ul Buffer, 0.5ul Primer Mix, 0.5ul RT Enzyme Mix, adds no enzyme water total to 10ul
System under the conditions of 37 DEG C, carries out the reverse transcription reaction of 15min → under the conditions of 98 DEG C, carries out the enzyme inactivation reaction of 5min → anti-
After answering, take part carry out 10 × concentration dilution, each miRNA will a special reverse transcription primer, so each
The reverse transcription reaction of a miRNA all independently carries out,
5) cDNA product carries out quantitative fluorescent PCR reaction
According to RR820A takaraPremix Ex TaqTMII (TAKARA, RR820A) is corresponding not to walk to carry out, institute
There is reaction to be 3 multiple holes, 10ulPremix, each 0.5ul of upstream and downstream primer, 1ul template sample, aqua sterilisa 8ul, altogether
20ul reaction system is counted, PCR reaction condition is 50 DEG C, 2min, 95 DEG C, 10min；95 DEG C, 1min；95 DEG C, 15s, 60 DEG C,
30s, 40 circulations, terminal acquires fluorescence, by expression quantity of the available each miRNA of the reaction in different samples, into
And subsequent analysis is carried out,
6) data are analyzed
Fluorescence quantitative PCR detection miRNA relative expression quantity, final result is with 2-△△CtAnalysis, using professional mapping software
Graphpad Prism7 is mapped and is statisticallyd analyze, and when P < 0.05, there is significant difference, analysis content is miRNA in morning
The individual difference analysis expressed in phase liver cancer patient, patient with chronic HBV and healthy population.
It is another object of the present invention to provide the preparation methods of excretion body miRNA-455-3p, comprising the following steps:
1) the excretion body in blood plasma is isolated；
2) miRNA in excretion body is extracted；
3) quantitative fluorescent PCR reaction detection miRNA.
The beneficial effects of the present invention are: it is successfully separated and identifies human plasma excretion body.Early liver cancer patients blood plasma's excretion
The level of miRNA-455-3p dramatically increases (P < 0.05) compared with chronic HBV infection group and healthy control group in body.This discovery is very
Good compensates for the status for lacking early diagnosing cancer of liver marker, and has wide market application prospect.Meanwhile passing through real-time fluorescence
Detection of the quantitative PCR technique to excretion body microRNA level in early liver cancer blood plasma, it is quantitative accurate, relative to chip technology,
Molecular hybridization or high throughput sequencing technologies, this method is simple and quick, economical and practical and carry out convenient for clinical.
Detailed description of the invention:
Figure 1A scattered signal figure
Figure 1B grain size distribution
Fig. 1 C average size
Fig. 1 is the particle size and concentration of the excretion body of nanometer flow cytometer detection；
Fig. 2 is to identify extracted blood plasma excretion body with the method for transmission electron microscope；
Fig. 3 be with after excretion body differential protein (CD9) label in the case where Lycra laser co-focusing superelevation point becomes fluorescence microscope
The excretion body observed；
Fig. 4 be with Aminis imaging streaming observe containing liver cancer differential protein (glypican,
Glypican-3 excretion body (CD9 label))；
Fig. 5 is in healthy control group (A1), chronic hepatitis B group (B2) and early liver cancer group (C3) blood plasma excretion body
The expression of miRNA-455-3p, note: liver cancer group is compared with other two groups, * P < 0.05.
Specific embodiment
In order to deepen the understanding of the present invention, the present invention is further described below in conjunction with embodiment and attached drawing, it should
The examples are only for explaining the invention, does not restrict the protection scope of the present invention.
Embodiment 1
One, research object: the morning in July, 2016 in December, 2017 Beijing YouAn Hospital, Capital Medical University is chosen
Phase liver cancer patient 22 are set as study group.Wherein male 10, female 12.Age 41~76 years old, average (60.1 ± 10.6) year.
Early liver cancer diagnostic criteria is defended planning commission according to country and is printed and distributed " primary carcinoma of liver diagnosis and treatment specification (version in 2017) ", all to leave and take blood plasma
Liver cancer patient gross tumor volume≤5cm and be single-shot tumour.Chronic HBV infection group patient 25, wherein male 13, female 12
Example, the age 40~65 years old, average (52.2 ± 9.7) year.The diagnostic criteria of chronic hepatitis B is according to Chinese Medical Association's hepatopathy
Chinese Medical Association, branch, which infects sick credit, can issue " chronic hepatitis B diagnostic criteria (version in 2015) ".Same period hospital is faced
Make a definite diagnosis healthy volunteer 26 of inspection center are set as control group, wherein male 13, female 14, the age 36~69 years old, average
(61.3 ± 9.3) year.This research obtains Hospital Ethical Committee's approval.
Study group and healthy control group.Chronic HBV infection group no difference of science of statistics (P > 0.05) between gender, age；
| Group | Number of cases | Age (year) | Male |
| Healthy control group | 26 | 61.3±9.3 | 12 |
| Chronic HBV group | 25 | 52.2±9.7 | 13 |
| Early liver cancer group | 22 | 60.1±10.6 | 15 |
| P value | 0.69 | 0.59 | 0.61 |
Two, experimentation
1. the preparation of plasma sample
EDTA anti-coagulants is added in heparin tube, after having acquired blood, heparin tube is slowly mixed by inversion, the whole blood of mixing
4 DEG C 1,000-2,000 × g is centrifuged 5-10min, and upper layer yellow translucent liquid is blood plasma to be collected, and pastes when blood plasma is sucked out
The page gradually inhale down, be sure not be sucked out cell component；The blood plasma being collected into can be directly used for subsequent experimental or dispense -80 DEG C
Refrigerator saves.
2. the extraction of excretion body
By blood plasma (fresh sample or the blood plasma for being stored in -80 degree melt on ice) room temperature, 2,000 × g is centrifuged 20min,
Remove residual cell and fragment；Supernatant is shifted into new centrifuge tube, is careful not to be drawn onto bottom precipitation；Room temperature, 10,000 × g
It is centrifuged 20min, removes residual fragment；With pipettor transfer supernatant into new centrifuge tube (be careful not to be drawn onto bottom precipitation and
Remaining surplus liquid), 500ul 1 × PBS and 300ulVEX Exosome Isolation Reagent is added in 1ml plasma sample
Mixed liquor is put in 2-8 DEG C of standing 30min after mixing vertically and is incubated for by solution, room temperature 10, and 000 × g centrifugation 5min is gone
Clearly, then room temperature 10,000 × g are centrifuged 30s, absorb residual liquid with pipettor, it is heavy that blood plasma excretion body is present in bottom of the tube
In shallow lake.The partial size for the blood plasma excretion body that this experiment is obtained by this method is simultaneously inhomogenous, and nanometer flow cytometer showed shows average grain diameter
It is mainly distributed on 62nm nearby (Fig. 1)；Excretion body is generally circular, partially has recess, partial size between 30-150nm (Fig. 2)；
Express the specificity marker molecule (Fig. 2 and Fig. 3) of CD9 excretion body.The excretion body extracted in early liver cancer patient group contains liver
The marker protein of cancer specificity --- glypican (Glypican-3, GPC3), so for special outer of liver cancer
Body (Fig. 3) is secreted, illustrates to be successfully separated human plasma excretion body, and the excretion body of liver cancer-specific can be separated to.
3. the extraction of excretion body miRNA
Excretion body miRNA, which is extracted, uses kit (TIANGEN BIOTECH (BEIJING) CO., LTD).Primary operational step
It is rapid as follows: 1. to take excretion body, 1ml Trizol is added and is sufficiently homogenized, oscillator oscillation or pipettor suction are beaten and mixed for several times.Room temperature
5min is stood, so that nucleic acid-protein compound is kept completely separate.2. 4 DEG C of 12,000rpm (13,400 × g) are centrifuged 5min, take
Clearly, it is transferred in the new centrifuge tube without RNase.3. 200 μ l chloroforms are added, pipe lid is covered, 15sec, room temperature are acutely vibrated
Place 5min.4. 4 DEG C of 12,000rpm (13,400 × g) are centrifuged 15min, sample can be divided into three layers: the organic phase of yellow, intermediate
Layer and colourless water phase, for RNA mainly in water phase, the volume of water phase is about the 50% of lysate MZ reagent used.Water phase is turned
It moves on in new pipe, carries out next step operation.5. measuring the volume of transfer liquid, it is slowly added to the anhydrous second of 0.43 times of transfer liquid product
Alcohol (such as: the transfer liquid of 500 μ l adds 215 μ l dehydrated alcohols) mixes.Obtained solution and precipitating are transferred to together to adsorption column
In miRspin, room temperature 12,000rpm (13,400 × g) is centrifuged 30sec, if once complete soln and mixture cannot be added
It into adsorption column miRspin, is please transferred in two times, is discarded after centrifugation to adsorption column miRspin, retain efflux.6. measuring stream
The volume of liquid out, being slowly added to 0.75 times of effluent volume of dehydrated alcohol, (such as: the efflux of 700 μ l adds the 525 anhydrous second of μ l
Alcohol), it mixes.Obtained solution and precipitating are transferred to together in adsorption column miRelute, room temperature 12,000rpm (13,400 × g)
It is centrifuged 30sec.7. 500 μ l protein liquid removal MRD are added into adsorption column miRelute, it is stored at room temperature 2min, room temperature 12,000rpm
(13,400 × g) is centrifuged 30sec, abandons waste liquid.8. 500 μ l rinsing liquid RW are added into adsorption column miRelute, it is stored at room temperature
2min, 12,000 rpm (13,400 × g) of room temperature are centrifuged 30sec, abandon waste liquid.9. repetitive operation step 7.10. by adsorption column
MiRelute is put into 2ml collecting pipe, room temperature 12, and 000rpm (13,400 × g) is centrifuged 1min, removes residual liquid.It will absorption
Column miRelute is transferred in a new RNase-Free 1.5ml centrifuge tube, adds 15 μ l RNase-FreeddH2O, and room temperature is put
2min, room temperature 12 are set, 000rpm (13,400 × g) is centrifuged 2min.
4.cDNA synthesis
Take 20ngRNA, based on ReverTra Ace qPCR RT Kit:FSQ-101, TOYOBO Reverse Transcriptase kit into
Row.5 × RT of 2ul Buffer, 0.5ul Primer Mix, 0.5ul RT Enzyme Mix is added, adds no enzyme water total to 10ul
System under the conditions of 37 DEG C, carries out the reverse transcription reaction of 15min → under the conditions of 98 DEG C, carries out the enzyme inactivation reaction of 5min → anti-
After answering, part is taken to carry out 10 × concentration dilution.Each miRNA will a special reverse transcription primer, so each
The reverse transcription reaction of a miRNA all independently carries out.
5.cDNA product carries out quantitative fluorescent PCR reaction
According to RR820A takaraPremix Ex TaqTMII (TAKARA, RR820A) is corresponding not to walk to carry out, institute
There is reaction to be 3 multiple holes, 10ulPremix, each 0.5ul of upstream and downstream primer, 1ul template sample, aqua sterilisa 8ul, altogether
Count 20ul reaction system.PCR reaction condition is 50 DEG C, 2min, 95 DEG C, 10min；95 DEG C, 1min；95 DEG C, 15s, 60 DEG C,
30s, 40 circulations, terminal acquires fluorescence, by expression quantity of the available each miRNA of the reaction in different samples, into
And carry out subsequent analysis.
6. data are analyzed
Fluorescence quantitative PCR detection miRNA relative expression quantity, final result is with 2-△△CtAnalysis, using professional mapping software
Graphpad Prism7 is mapped and is statisticallyd analyze, and when P < 0.05, there is significant difference.Analyzing content is miRNA in morning
The individual difference analysis expressed in phase liver cancer patient, patient with chronic HBV and healthy population.Early liver cancer patient group miRNA-455-
The expression of 3p increases (P < 0.05) than more significant with patient with chronic HBV's group and healthy control group.Therefore miRNA-455-3p can make
For the marker of diagnosing cancer of liver (early stage).
Claims (10)
1. a kind of excretion body miRNA-455-3p for diagnosing liver cancer.
2. excretion body miRNA-455-3p according to claim 1, which is characterized in that the sequence of the miRNA-455-3p
For 5 '-GCAGUCCAUGGGCAUAUACAC-3 '.
3. application of the excretion body miRNA-455-3p described in claim 1 in terms of diagnosing liver cancer.
4. application of the excretion body miRNA-455-3p described in claim 1 in terms of diagnosing early liver cancer.
5. excretion body miRNA-455-3p described in claim 1 answering in terms of diagnosing early liver cancer as tumor marker
With.
6. excretion body miRNA-455-3p answering in the product for preparing early liver cancer diagnostic marker described in claim 1
With.
7. excretion body miRNA-455-3p described in claim 1 is preparing the application in diagnosing cancer of liver kit.
8. according to application described in claims requirement 7, which is characterized in that the liver cancer is early liver cancer.
9. the preparation method of excretion body miRNA-455-3p described in claim 1 mainly includes following 3 parts:
1) the excretion body in separated plasma；
2) miRNA in excretion body is extracted；
3) quantitative fluorescent PCR reaction detection miRNA.
10. application according to claim 5 or 6, which is characterized in that
1) preparation of plasma sample
EDTA anti-coagulants is added in heparin tube, after having acquired blood, heparin tube is slowly mixed by inversion, 4 DEG C of the whole blood of mixing
1,000-2,000 × g are centrifuged 5-10min, and upper layer yellow translucent liquid is blood plasma to be collected, close to page when blood plasma is sucked out
Face is gradually inhaled down, is sure not that cell component is sucked out；The blood plasma being collected into can be directly used for subsequent experimental or dispense -80 DEG C of refrigerators
It saves,
2) extraction of excretion body
By blood plasma room temperature, 2,000 × g is centrifuged 20min, removes residual cell and fragment；Supernatant is shifted into new centrifuge tube, note
Meaning not be drawn onto bottom precipitation；Room temperature, 10,000 × g are centrifuged 20min, remove residual fragment；Supernatant is shifted to newly with pipettor
Centrifuge tube in, 1ml plasma sample, be added 500ul 1 × PBS and 300ulVEX Exosome Isolation Reagent it is molten
Mixed liquor is put in 2-8 DEG C of standing 30min after mixing vertically and is incubated for by liquid, room temperature 10, and 000 × g centrifugation 5min removes supernatant,
Then room temperature 10,000 × g are centrifuged 30s, absorb residual liquid with pipettor, and blood plasma excretion body is present in bottom of the tube precipitating,
3) extraction of excretion body miRNA
Excretion body miRNA, which is extracted, uses kit, and main operational steps are as follows: 1. taking excretion body, it is abundant that 1ml Trizol is added
Homogenate, oscillator oscillation or pipettor suction are beaten and are mixed for several times, and 5min is stored at room temperature, so that nucleic acid-protein compound is kept completely separate,
2. 4 DEG C of 12,000rpm (13,400 × g) are centrifuged 5min, supernatant is taken, is transferred in the new centrifuge tube without RNase, is 3. added
200 μ l chloroforms, cover pipe lid, acutely vibrate 15sec, be placed at room temperature for 5min, 4. 4 DEG C of 12,000rpm (13,400 × g) centrifugations
15min, sample can be divided into three layers: the organic phase of yellow, middle layer and colourless water phase, and RNA is mainly in water phase, the body of water phase
The 50% of product lysate MZ reagent about used, water phase is transferred in new pipe, carries out next step operation, 5. measure transfer liquid
Volume is slowly added to the dehydrated alcohol of 0.43 times of transfer liquid product, mixes, obtained solution and precipitating are transferred to together to absorption
In column miRspin, room temperature 12,000rpm (13,400 × g) is centrifuged 30sec, if cannot once add complete soln and mixture
Enter into adsorption column miRspin, be please transferred in two times, is discarded after centrifugation to adsorption column miRspin, retain efflux, 6. measure
The volume of efflux is slowly added to 0.75 times of effluent volume of dehydrated alcohol, mixes, and obtained solution and precipitating are turned together
Enter in adsorption column miRelute, room temperature 12,000rpm (13,400 × g) is centrifuged 30sec, is 7. added into adsorption column miRelute
500 μ l protein liquid removal MRD are stored at room temperature 2min, room temperature 12, and 000rpm (13,400 × g) is centrifuged 30sec, waste liquid are abandoned, 8. to suction
500 μ l rinsing liquid RW are added in attached column miRelute, are stored at room temperature 2min, room temperature 12,000rpm (13,400 × g) centrifugation
30sec abandons waste liquid, and 9. adsorption column miRelute, is 10. put into 2ml collecting pipe by repetitive operation step 7, room temperature 12,000rpm
(13,400 × g) is centrifuged 1min, removes residual liquid, adsorption column miRelute is transferred to a new RNase-Free 1.5ml
In centrifuge tube, add 15 μ l RNase-FreeddH2O, is placed at room temperature for 2min, room temperature 12,000rpm (13,400 × g) centrifugation
2min,
4) cDNA is synthesized
20ngRNA is taken, is carried out, is added based on ReverTra Ace qPCR RT Kit:FSQ-101, TOYOBO Reverse Transcriptase kit
Enter 5 × RT of 2ul Buffer, 0.5ul Primer Mix, 0.5ul RT Enzyme Mix, add no enzyme water to 10ul total system,
Under the conditions of 37 DEG C, the reverse transcription reaction of 15min → under the conditions of 98 DEG C is carried out, the enzyme inactivation reaction → reaction for carrying out 5min terminates
Afterwards, take part carry out 10 × concentration dilution, each miRNA will a special reverse transcription primer, so each miRNA
Reverse transcription reaction all independently carry out,
5) cDNA product carries out quantitative fluorescent PCR reaction
According to RR820A takaraPremix Ex TaqTMII (TAKARA, RR820A) is corresponding not to walk to carry out, all anti-
3 multiple holes, 10ul should bePremix, each 0.5ul of upstream and downstream primer, 1ul template sample, aqua sterilisa 8ul amount to
20ul reaction system, PCR reaction condition are 50 DEG C, 2min, 95 DEG C, 10min；95 DEG C, 1min；95 DEG C, 15s, 60 DEG C, 30s, 40
A circulation, terminal acquire fluorescence, by expression quantity of the available each miRNA of the reaction in different samples, and then carry out
Subsequent analysis,
6) data are analyzed
Fluorescence quantitative PCR detection miRNA relative expression quantity, final result is with 2-△△CtAnalysis, using professional mapping software
Graphpad Prism7 is mapped and is statisticallyd analyze, and when P < 0.05, there is significant difference, analysis content is miRNA in morning
The individual difference analysis expressed in phase liver cancer patient, patient with chronic HBV and healthy population.
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