CN110205374A - Detect primer, kit, detection method and its application of miR-34a gene expression amount in blood platelet - Google Patents
Detect primer, kit, detection method and its application of miR-34a gene expression amount in blood platelet Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of primers of miR-34a gene expression amount in detection blood platelet, the primer is miR-34a specific primer, the reverse primer of the specific primer as shown in SEQ ID NO:1, forward primer as shown in SEQ ID NO:2, reverse primer is as shown in SEQ ID NO:3.The invention also discloses the applications of the kit of miR-34a gene expression amount, detection method and above-mentioned primer and kit in a kind of detection blood platelet.The primer and kit can be used for detecting the expression quantity of miR-34a gene related with acute myocardial infarction AMI.The present invention quick and precisely, it is easy to operate.
Description
Technical field
The present invention relates to the primer of miR-34a gene expression amount in a kind of detection blood platelet, kit, detection method and its
Using belonging to gene expression amount detection technique field.
Background technique
As the living standard of the people is gradually increased, coronary artery disease just like has become seriously threatens people's body
The principal element of Health and Living quality.In 60 years old or more old man, coronary artery disease is that dead first is caused to lure
Cause, and the nowadays morbidity crowd of coronary artery disease more rejuvenation.The World Health Organization declares the whole world by coronary artery disease
Sick influence number will reach 82,000,000 people in the year two thousand twenty.In numerous disease phenotypes of coronary artery disease, Acute myocardial stalk
Extremely (abbreviation acute myocardial infarction) disease incidence highest and consequence most serious, every year because acute myocardial infarction death number is more than 7,000,000 people.It is anxious
Property heart infarction high incidence and high lethality rate be largely because the diagnosis to the disease not in time and also lack effectively it is special
Diagnosis index.The main pathogenesis of acute myocardial infarction AMI is to rupture secondary thrombus shape by Coronary Atherosclerotic Plaque
At, and then blocked coronary arteries lumen, lead to myocardial ischemia and necrosis.In the early stage that coronary atherosclerosis occurs, move
Blood platelet can be adsorbed by the vascular endothelial cell that inflammatory reaction activates on astillen, lead to blood platelet in the aggregation at the position and swashed
It is living.The blood platelet of activation can secrete a large amount of rush cell adhesion factor and proinflammatory cell factor, enhance the inflammation at the position
It reacts and then patch is accelerated to be formed.Under the collective effect of endothelial cell, immunocyte and blood platelet, patch can gradually aggravate shape
At the stabilization patch or high risk Vulnerable plaque that can cause Stable angina pectorsis.When patient meets with some acute infections or stimulation
Afterwards, the inflammatory reaction at Vulnerable plaque position can also be increase accordingly, and the endothelial cell around patch can go out because of inflammatory reaction at this time
Existing a degree of apoptosis.After there is apoptosis in endothelial cell around the plaque in weaker endodermis, interior leather wallet
Plaque in layer will be exposed in blood flow, this process is exactly plaque rupture.Once the plaque quilt in interlayer
It is exposed in blood flow, blood platelet will be integrated on patch and be activated by a variety of receptor systems.The blood platelet of activation then can
A large amount of active materials are discharged into ambient enviroment, to activate more blood platelets and immunocyte, and cause blood platelet in spot
Block rent is largely assembled, and thrombus is gradually formed.Draw it can be seen that being formed from Coronary Atherosclerotic Plaque to plaque rupture
During the entire process of playing acute myocardial infarction, blood platelet has all played very crucial effect.Therefore, front and back occurs for research acute myocardial infarction
The variation of the platelet of different times is prevented and treated for finding Accurate Prediction and diagnosing the biology mark object of acute myocardial infarction
Acute myocardial infarction is all of great significance.
Traditional detection means are to detect to the content of Blood Center flesh troponin, but actual conditions are acute
The 3-4 hours dirty specific troponins of Blood Center just can be increased significantly after the onset of heart infarction.Clinically develop a kind of high sensitivity
Degree method detects cardiac troponin, but it sacrifices accuracy while improving susceptibility: this high sensitive
Detection method can also detect very high cardiac troponin signal in non-acute heart infarction patient.Therefore there is an urgent need to find one
Kind can quick and precisely judge that acute myocardial infarction even predicts the biological indicator of acute myocardial infarction morbidity.
It is mainly mediated by miRNA in view of miRNA and its gene expression regulation very rich is contained in blood platelet, and blood is small
Plate takes part in patch shape at the whole process for causing embolism to lead to acute myocardial infarction to plate rupture, Wo Mentong from atherosclerosis
The research discovery for spending early period: miRNA express spectra can occur acutely to change in the platelet cell of acute myocardial infarction patient, and this
A little miRNA can not only regulate and control the gene expression of blood platelet itself, can be also discharged by excretion body in blood circulation thin by other
Born of the same parents absorb to adjust the gene expression of target cell.We are by comparing in the blood platelet of acute myocardial infarction patient and Healthy People simultaneously
MiRNA express spectra, searches out the miRNA that expression significantly changes, i.e. miR-34a, and in acute myocardial infarction patient blood sample and small
It is verified in mouse acute myocardial infarction model, experimental result shows that miR-34a has the diagnosis biological marker as acute myocardial infarction
The ability of object.
Summary of the invention
One of the objects of the present invention is to provide primer, the reagents of miR-34a gene expression amount in a kind of detection blood platelet
Box, detection method and its application, quick and precisely, easy to operate, to solve to carry out acute myocardial infarction AMI anticipation inspection in the prior art
The problem of ranging sequence is cumbersome, the period is long, somewhat expensive.
To achieve the above object, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of primer of miR-34a gene expression amount in detection blood platelet, the primer is miR-
34a specific primer, the reverse primer of the specific primer is as shown in SEQ ID NO:1, forward primer such as SEQ ID NO:2
Shown, reverse primer is as shown in SEQ ID NO:3.
On the other hand, the present invention provides a kind of kits of miR-34a gene expression amount in detection blood platelet, comprising upper
State the primer of miR-34a gene expression amount in detection blood platelet.
Further, the kit further includes 2 × SYBR Green fluorescent quantitation premixed liquid, and it is poly- that it includes Taq DNA
Synthase, PCR buffer, dNTP, SYBR Green dyestuff, ROX internal reference dyestuff and ddH2O.
Further, the kit further includes reference gene primer, wherein the reference gene is house-keeping gene U6, institute
The forward primer of house-keeping gene U6 is stated as shown in SEQ ID NO:4, reverse primer is as shown in SEQ ID NO:5.
On the other hand, the present invention also provides a kind of methods of miR-34a gene expression amount in detection blood platelet, including with
Lower step:
(1) using in miRNA separating kit extracting patients of acute myocardial infarction blood platelet or blood platelet excretion body sample
RNA；
(2) cDNA library of the RNA sample obtained using reverse transcription reagent box preparation step (1)；
(3) fluorescent quantitative PCR is carried out as template using the cDNA that step (2) obtains, detects blood platelet and blood platelet excretion body
MiR-34a expression quantity in sample；Wherein the primer of fluorescent quantitative PCR includes miR-34a specific primer, the specificity
The reverse primer of primer as shown in SEQ ID NO:1, forward primer is as shown in SEQ ID NO:2, reverse primer such as SEQ ID
Shown in NO:3.
Further, the reaction system of the fluorescent quantitative PCR are as follows:
2.5 μ L of miR-34a specific primer
2 × SYBR Green fluorescent quantitation premixed liquid, 12.5 μ L
cDNA 4 μL
ddH2O 6 μL
Further, detection miR-34a expression quantity is described using relative quantification method using house-keeping gene U6 as internal reference in step (3)
The forward primer of house-keeping gene U6 is as shown in SEQ ID NO:4, and reverse primer is as shown in SEQ ID NO:5.
On the other hand, the present invention also provides the primers of miR-34a gene expression amount in above-mentioned detection blood platelet to prepare
Application in detection or diagnosis acute myocardial infarction AMI reagent.
On the other hand, the present invention also provides the kits of miR-34a gene expression amount in above-mentioned detection blood platelet to examine
Survey the application in miR-34a gene expression amount related with acute myocardial infarction AMI.
Present invention advantageous effects achieved: the present invention quick and precisely, it is easy to operate, solve in the prior art into
The problem of row acute myocardial infarction AMI anticipation detection program is cumbersome, the period is long, somewhat expensive.
Detailed description of the invention
Fig. 1 is testing result of the detection kit provided in an embodiment of the present invention to miR-34a expression quantity in blood platelet
Figure；
Fig. 2 is testing result of the detection kit provided in an embodiment of the present invention to miR-34a expression quantity in blood plasma excretion body
Figure；
Fig. 3 is detection knot of the detection kit provided in an embodiment of the present invention to miR-34a expression quantity in blood platelet excretion body
Fruit figure.
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention
Technical solution, and not intended to limit the protection scope of the present invention.The implementation condition used in the following example can root
Further adjustment is done according to condition proposed by manufacturer, the implementation condition being not specified is usually that normal condition such as molecular cloning is real
Test guide, the third edition, Science Press, condition described in 2002.
Sample and method
It is punctured by the antecubital vein on seat, fresh human experimenter's venous blood sample is collected in standardization.Whole blood (20 mL) is set
In the test tube containing EDTA, it is centrifuged (at 4 DEG C, 1200 g, 10 minutes).Supernatant is collected, be centrifuged (at 4 DEG C, 12000 grams, 10 points
Clock), take blood plasma.Take 5mL blood plasma rapidly extracting RNA.The detection of PCR product relative quantification: being internal reference to target using house-keeping gene U6
Gene is normalized, using the variation of formula RQ=2- △ △ CT calculation expression amount multiple.
Embodiment
The present invention relates to a kind of kits of miR-34a gene expression amount in detection blood platelet, include:
(1) miR-34a detection reagent, it includes miR-34a specific primer, the reverse primer of the specific primer such as SEQ
Shown in ID NO:1, forward primer is as shown in SEQ ID NO:2, and reverse primer is as shown in SEQ ID NO:3；
(2) 2 × SYBR Green fluorescent quantitation premixed liquid (archaeal dna polymerase containing Taq, PCR buffer, dNTP, SYBR Green
Dyestuff, ROX internal reference dyestuff and ddH2O)
(3) house-keeping gene U6 specific primer, the forward primer of the specific primer reversely draw as shown in SEQ ID NO:4
Object is as shown in SEQ ID NO:5.
The content (3) of the kit be miR-34a expression reference standards, convenient for user to testing result into
The control of row quality.
It is the blood of acute myocardial infarction patient and 30 clinical diagnosises without any different that 25 clinical diagnosises are collected in the present embodiment
Normal normal human blood, and therefrom extract microRNA(miRNA), it detects in sample to be tested and is expressed with the presence or absence of miR-34a
Amount, concrete operation step are as follows:
1. the extraction of RNA in blood platelet and blood platelet excretion body sample
Use the cell tissue mirVana of Thermo Fisher companyTMMiRNA separating kit (mirVanaTMmiRNA
Detection Kit) (article No.: AM1552) extracting acute myocardial infarction Platelet and blood platelet excretion body sample RNA.
2. prepared by the cDNA library of acute myocardial infarction AMI
Using QIAEGEN company reverse transcription reagent box (QuantiTect Reverse Transcription Kit, article No.:
205313) cDNA library for the RNA sample that preparation step 1 obtains.)
3. the preparation of PCR reaction system
PCR reaction system in the preferred embodiment of the present invention containing above-mentioned (1)-(3) number reagent is 25 μ L, can be according to
Following ratio is prepared:
(1) 2.5 μ L of number reagent (miR-34a specific primer)
(2) number 2 × SYBR Green fluorescent quantitation premixed liquid, 12.5 μ L
4 μ L of cDNA prepared by step 2
ddH2O 6 μL
The configuration of house-keeping gene U6 detection quality control system:
(3) 2.5 μ l of number reagent (U6 specific primer)
(2) number 2 × SYBR Green fluorescent quantitation premixed liquid, 12.5 μ L
4 μ L of cDNA prepared by step 2
ddH2O 6 μL
4. above-mentioned reaction system, which is put into PCR instrument, carries out PCR amplification, using house-keeping gene U6 as internal reference, using relative quantification method,
Detect the expression quantity of miR-34a in blood platelet and blood platelet excretion body sample.
25 acute myocardial infarction patients and the testing result of the blood clinical sample of 30 normal persons are as follows in the present embodiment:
MiR-34a expression is relative to normal in acute myocardial infarction Platelet, blood plasma excretion body and blood platelet excretion body
Have per capita and substantially change, wherein miR-34a expression quantity is remarkably reinforced relative to normal person, changes maximum in blood platelet, secondly
It is miR-34a expression quantity in blood platelet excretion body, and the expression quantity of miR-34a then changes minimum in blood plasma excretion body, result
As shown in Figures 1 to 3.Experimental result shows that miR-34a can be used as the diagnostic biomarkers of acute myocardial infarction.
The present invention is disclosed with preferred embodiment above, so it is not intended to limiting the invention, all to take equivalent replacement
Or the scheme technical solution obtained of equivalent transformation, it falls within the scope of protection of the present invention.
Sequence table
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Claims (9)
1. the primer of miR-34a gene expression amount in a kind of detection blood platelet, which is characterized in that the primer is miR-34a special
Specific primer, the reverse primer of the specific primer as shown in SEQ ID NO:1, forward primer as shown in SEQ ID NO:2,
Reverse primer is as shown in SEQ ID NO:3.
2. the kit of miR-34a gene expression amount in a kind of detection blood platelet, which is characterized in that comprising described in claim 1
Primer.
3. the kit of miR-34a gene expression amount in detection blood platelet according to claim 2, which is characterized in that also
Including 2 × SYBR Green fluorescent quantitation premixed liquid, it includes Taq archaeal dna polymerase, PCR buffer, dNTP, SYBR Green
Dyestuff, ROX internal reference dyestuff and ddH2O.
4. the kit of miR-34a gene expression amount in detection blood platelet according to claim 2, which is characterized in that also
Including reference gene primer, wherein the reference gene is house-keeping gene U6, the forward primer such as SEQ of the house-keeping gene U6
Shown in ID NO:4, reverse primer is as shown in SEQ ID NO:5.
5. a kind of method of miR-34a gene expression amount in detection blood platelet, which comprises the following steps:
(1) using in miRNA separating kit extracting patients of acute myocardial infarction blood platelet or blood platelet excretion body sample
RNA；
(2) cDNA library of the RNA sample obtained using reverse transcription reagent box preparation step (1)；
(3) fluorescent quantitative PCR is carried out as template using the cDNA that step (2) obtains, detects blood platelet and blood platelet excretion body
MiR-34a expression quantity in sample；Wherein the primer of fluorescent quantitative PCR includes miR-34a specific primer, the specificity
The reverse primer of primer as shown in SEQ ID NO:1, forward primer is as shown in SEQ ID NO:2, reverse primer such as SEQ ID
Shown in NO:3.
6. the method for miR-34a expression quantity in detection blood platelet according to claim 5, which is characterized in that the fluorescence
The reaction system of quantitative pcr amplification are as follows:
2.5 μ L of miR-34a specific primer
2 × SYBR Green fluorescent quantitation premixed liquid, 12.5 μ L
cDNA 4 μL
ddH2O 6 μL 。
7. the method for miR-34a expression quantity in detection blood platelet according to claim 5, which is characterized in that in step (3)
MiR-34a expression quantity is detected using house-keeping gene U6 as internal reference, using relative quantification method, the forward primer of the house-keeping gene U6 is such as
Shown in SEQ ID NO:4, reverse primer is as shown in SEQ ID NO:5.
8. the primer of miR-34a gene expression amount in preparation detection or diagnoses acute in detection blood platelet described in claim 1
Application in myocardial infarction reagent.
9. the described in any item kits for detecting miR-34a gene expression amount in blood platelets of claim 2-4 are in detection and suddenly
Application in the property related miR-34a gene expression amount of myocardial infarction.
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|---|---|---|---|---|
| US20120238619A1 (en) * | 2011-03-16 | 2012-09-20 | Miragen Therapeutics | Micro-rna for the regulation of cardiac apoptosis and contractile function |
| CN102791859A (en) * | 2009-12-15 | 2012-11-21 | 得克萨斯系统大学董事会 | Micro-RNA regulation in ischemia and ischemia-reperfusion injury |
| CN103484550A (en) * | 2013-09-30 | 2014-01-01 | 中国科学院上海微系统与信息技术研究所 | MicroRNA biological markers for early lung cancer diagnosis and application thereof |
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2019
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| CN102791859A (en) * | 2009-12-15 | 2012-11-21 | 得克萨斯系统大学董事会 | Micro-RNA regulation in ischemia and ischemia-reperfusion injury |
| US20120238619A1 (en) * | 2011-03-16 | 2012-09-20 | Miragen Therapeutics | Micro-rna for the regulation of cardiac apoptosis and contractile function |
| CN103484550A (en) * | 2013-09-30 | 2014-01-01 | 中国科学院上海微系统与信息技术研究所 | MicroRNA biological markers for early lung cancer diagnosis and application thereof |
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| 潘嘉林，等: "MicroRNA-34a在大鼠心肌梗死后的细胞凋亡中的作用", 《心电与循环》 * |
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