CN110229893A - For diagnosing miRNAs marker and its application of carotid artery atherosclerosis plaques - Google Patents
For diagnosing miRNAs marker and its application of carotid artery atherosclerosis plaques Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses for diagnosing carotid artery atherosclerosis plaques miRNAs marker and its application.MiR-21, miR-155, miR-221, miR-222 are significantly raised compared with healthy control group with the expression of cerebral infarction group, Vulnerable plaque group, stable patch group in Vulnerable plaque, miR-126 is substantially reduced in Vulnerable plaque with the more stable patch group of expression in cerebral infarction group, Vulnerable plaque group, and Vulnerable plaque is also substantially reduced with the expression of miR-126 in cerebral infarction group compared with Vulnerable plaque group.The growth of above-mentioned 5 kinds of miRNAs and patch, unstable, rupture evolution process is closely related, can be applied to the screening of assessment stabilization of carotid atherosclerosis plaque and high risk population of stroke.
Description
Technical field
The present invention relates to laboratory medicine technical fields, and in particular to one group of diagnosis mark for carotid artery atherosclerosis plaques
Will object miRNAs and its application.
Background technique
It is investigated and is shown according to Ministry of Public Health's Third National cause of death, cranial vascular disease has become Chinese's disease death
First reason, midbrain infarction account for about 75%, with high incidence, high relapse rate, high disability rate, high lethality rate, high take
The characteristics of with " five is high ", causes serious burden to personal, family and society.China is used for stroke medical expense every year and is up to
40000000000 yuan.Embolus caused by carotid artery atherosclerosis plaques rupture be the main reason for leading to cerebral infarction formed not only with patch
Size is related, and obviously related to plaque stability.In recent years, the research of carotid plaques stability is increasingly by weight
Depending on, how accurate evaluation plaque stability be Modern Medical Field research one of hot spot, identify and to intervene unstable neck dynamic
Arteries and veins plaque is very great for the prevention and treatment meaning of stroke.The embolus caused by unstable carotid plaques rupture, falling off is led
When causing cerebral infarction, the patch is the responsibility patch of this cerebral infarction at this time, also has scholar to be referred to as criminal's patch.Iconography pair
The judgement of responsibility patch needs specifically to combine clinical knowledge and particular patient, there is no unified criterion.Carotid atherosclerosis
The identification of patch (stablize patch, vulnerable plaque) is clinical at present mainly by means of imageological examination, as B ultrasound, high-resolution MRI,
DSA inspection etc., wherein ultrasound diagnosis is often preferred, and the inspection is convenient, noninvasive, cheap, but its stability is passed through vulnerable to examiner
It tests, the personal subjectivity such as technology influences；High-resolution MRI expensive (1000 yuan/time), long to vascular wall imaging time,
Efficiency is lower, is chiefly used in scientific research, it is difficult to meet the needs of clinical a large amount of patients；And DSA check price it is more expensive (4000 yuan/
It is secondary), operation is invasive, and cannot still assess Stability of Atherosclerotic Plaques situation very well, also should not be in follow up
It reuses.Moreover, there is no ideal imageological examination energy total evaluation entire patient's atherosclerosis situation at present.Cause
This, is if the biomarker for distinguishing different types of carotid artery atherosclerosis plaques can further be found from peripheral blood,
It will be with important clinical meaning.That it has many advantages, such as is convenient, noninvasive, stability is good, inspection is quick, checks convenient for follow-up, energy
Enough meets the needs of clinical " blowout " or " tsunami formula " carotid artery atherosclerosis plaques patient identification and Treatment monitoring.
As " microRNAs is as the mechanism for predicting atherothrombosis in the excretion corpusculum of blood platelet source for document
Research " it discloses blood platelet source excretion corpusculum miR-223, miR-339 and miR-21 and can be used as the mark of internal platelet activation
Will object, miR-223, miR-339 and miR-21 level increase related to prediction thrombosis.
Separately there is document " circulating plasma miRNA is worth community hypertension patients carotid atherosclerosis early prediction " open
The relative expression of has-miR-33, has-miR-125a-5p, has-miR-126, has-miR-145 and has-miR-146a
Level is between hypertensive patients carotid atherosclerosis and patient without carotid atherosclerosis and without carotid atherosclerosis
There is apparent differential expression.Wherein has-miR-33 and has-miR-146a obviously rises in the patient of carotid atherosclerosis
Height, and has-miR-125a-5p, has-miR-126 and has-miR-145 obviously drop in the patient of carotid atherosclerosis
It is low.And the change of the relative expression levels of this five kinds of miRNA is obviously related to degree of carotid in follow-up, mentions
Show that the change of the relative expression levels of five kinds of miRNA can be used as clinically early prediction hypertensive patient and Atherosclerosis occurs
The hematological indices of change.
MiRNA is the non-coding regulatory RNA of a kind of length about 18-24nt, is had well-conserved.Their water after transcription
It is flat to play the role of retroregulation by inhibiting translation or degradation to target mRNA.MiRNA participates in various tune in vital movement
Section approach, such as ontogeny, cell Proliferation, apoptosis, fat metabolism vital movement.During Vascular Biology, such as blood vessel
It generates, reparation, endothelial cell proliferation etc. after vascular endothelial cell damage, today there is also many researchs to confirm that miRNA performance is focused on
The effect wanted such as has been knocked in the mouse model of miR-145 and miR-143 and has observed, vascular smooth muscle cells, which are in, does not divide
Change state can not form normal blood vessel endothelium, influence new vessels and formed and repaired after endothelial injuries；Divide in rat model
Qiao Chu not be after miR-21 or miR-221 and miR-222, it was similarly observed that vascular endothelial cell proliferation is suppressed, Apoptosis adds
Speed；Meanwhile also studies have found that miR-103a is by inhibiting anti-angiogenesis gene GAX and HOXA5 that can promote new vessels shape
At；Rat carotid artery ligation model also demonstrates the endometrial hyperplasia reparation after miRNA takes part in injury of blood vessel；These results of study
Prompt miRNA is newborn to blood vessel endothelium, repair after inner film injury, new vessels are formed etc. plays crucial regulating and controlling effect.Therefore,
MiRNA and a series of vascular conditions, as Cardial or cerebral vascular diseases have close relationship.There is related scholar to demonstrate,prove in recent years
Real function of the miRNA by regulation endothelial cell, vascular smooth muscle cells, macrophage etc., participates in a variety of cardiovascular diseases
Pathological development process, including atherosclerosis, heart failure, myocardial infarction etc..In the difference progress of atherosclerosis
Stage, the level of specific miRNA occur corresponding change and by adjusting certain specific accesses, and such as cholesterol metabolic participates in
The formation of patch.
Although miRNA has preliminary research, system research inhomogeneity as the biomarker of atherosclerosis
The related miRNA biomarker of type patch (stablizing patch, rapid wear, responsibility patch) has not been reported.
The present invention is based on the different types of features of the plate of carotid atherosclerosis, disclose one group for diagnosing patch class
The miRNA marker and application of type carotid atherosclerosis.
Summary of the invention
To solve the above problems, firstly, the present invention provides one group for diagnosing the primer sets of carotid artery atherosclerosis plaques,
Primer particular sequence is as shown in table 1:
Table 1
Secondly, the present invention provides one group for diagnosing the miRNAs markers of carotid artery atherosclerosis plaques: miR-21,
miR-126,miR-155,miR-221,miR-222；
Specifically: miR-21, miR-155, miR-221, miR-222 are in Vulnerable plaque with cerebral infarction group, unstable plaque
Block group, the expression for stablizing patch group are significantly raised compared with healthy control group, and statistically significant difference (is P=
0.000).MiR-126 is bright with the more stable patch group of expression in cerebral infarction group, Vulnerable plaque group in Vulnerable plaque
Aobvious to reduce, difference is statistically significant (being P=0.000), and Vulnerable plaque is with the table of miR-126 in cerebral infarction group
It is also substantially reduced up to level compared with Vulnerable plaque group.
Again, the present invention provides a kind of method using miRNAs diagnosis carotid artery atherosclerosis plaques, the specific steps are as follows:
1. collecting Carotid Artery Atherosclerotic Patients and corresponding healthy serum sample；
2. Carotid Artery Atherosclerotic Patients and corresponding healthy serum Total RNAs extraction；
(1) serum sample for obtaining step 1 is transferred to centrifuge tube, and addition Trizol is placed in vortex concussion instrument and is acutely vortexed
Then 10-40s is stored at room temperature 2-10min, crack nucleoprotein complex sufficiently, and lysate is made；
(2) chloroform is added in the lysate made from step (1), acutely shakes 5-30s, be stored at room temperature 2-6min, obtains mixed
Close object A；
Wherein, the volume ratio of serum, Trizol and chloroform is 1:3:1；
(3) open in advance centrifuge make its it is pre- be cooled to 4 DEG C, by mixture A made from step (2), 12000g is centrifuged 10-
25min；
(4) it is transferred to new centrifuge tube with the supernatant fluid after pipettor aspiration step (3) centrifugation, 0.6~1 times of body is added
Long-pending isopropanol mixes, and the sample of mixing is incubated for 5-20min under the conditions of 15-20 DEG C, precipitates RNA；
(5) by the sample after precipitating in step (4), 12000g, abandons supernatant after 5min centrifugation, adds 0.5-2ml 75% by 4 DEG C
Ethyl alcohol cleaning precipitating, 7500g, 4 DEG C, 5min is centrifuged to obtain RNA crude product；
(6) the RNA crude product in step (5) is dried into precipitating, adds solvent dissolution and precipitates and save；
Preferably, the solvent in step 2- (6) is 20 μ l DEPC water；
3. detecting the content and quality of serum sample RNA；
4. design prepares specificity primer, specific primer sequence is as described in Table 1；
The synthesis of first chain of 5.cDNA:
It is added according to following systems in the reaction tube of RNase-free, is put with centrifuge letter from the drop on wall after mixing
It is reacted into PCR instrument；
Reaction condition setting: 42-50 DEG C, 45min；70 DEG C, 15min；
Shown in reaction system such as table 2:
Table 2
6.PCR amplified reaction:
Reagent is added in reaction vessel according to following systems, after mixing with centrifuge letter from, make dripless on wall, then into
Row RT-q PCR reaction, RT-qPCR agents useful for same are as shown in table 3:
Table 3
Reaction condition setting: 95 DEG C of 10min；Subsequently into 95 DEG C of 15s, the circulation (totally 40 times) of 60 DEG C of 1min.
7. data are analyzed
(1) relative quantification is carried out using reference gene as standard, existing reference gene 5S RNA carries out target miRNAs
Normalized；
(2) pass through relative expression quantity of the fluorescence quantitative PCR detection target miRNAs in different samples；
Specifically, the formula of the variation of expression quantity multiple are as follows:
△ Ct (normal serum)=Ct (normal serum miRNA)-Ct (reference gene), △ Ct (carotid atherosclerosis blood
Clearly)=Ct (carotid atherosclerosis serum miRNA)-Ct (reference gene), RQ (normal/carotid atherosclerosis)=2-△ Ct
(carotid atherosclerosis)-△ Ct (normal)；
Wherein, RQ represents relative expression quantity (Relative Quantitation, RQ)；
Ct (miRNA) and Ct (reference gene) respectively represents the target miRNAs that fluorogenic quantitative detection arrives and reference gene
Ct value；
(3) statistical analysis is carried out to the experimental data that quantitative fluorescent PCR obtains, and passes through one-way analysis of variance target
Differential expression analysis of the miRNAs in different samples, when bilateral P value < 0.05, it is believed that the differential expression of the two is in statistics
It is upper that there is significant difference；When bilateral P value < 0.01, it is believed that the differential expression of the two statistically has extremely significant property poor
It is different.
Finally, the present invention provides a kind of above-mentioned miRNAs marker of utilization and method in diagnosis carotid artery atherosclerosis plaques
In application.
Detailed description of the invention
Fig. 1 is miR-21, miR-126, miR-155, miR-221, miR-222 expression of 4 group objects in embodiment 7
Comparison result figure；
Fig. 2 is miRNAs expression and blood lipid 4 horizontal correlation results figures in embodiment 7.
Specific embodiment
A specific embodiment of the invention is described with attached drawing combined with specific embodiments below, in order to preferably manage
The solution present invention.
Key instrument of the present invention is as shown in table 4:
Table 4
The serum sample of 1 Carotid Artery Atherosclerotic Patients of embodiment and corresponding normal healthy controls is collected and is arranged
Cerebral infarction group patient 35 that in September, 2016 in March, 2017 our hospital's Neurology is gone to a doctor are chosen, 24 male, female
11, year at age 50~84 (69.3 ± 10.6)；It chooses same time medical center and is confirmed the existence of arteria carotis not through Doppler's color ultrasound
Stablize plaque patients (Vulnerable plaque group) 41, male 24, female 17, the age 45~90 (66.0 ± 11.2) year；Stablize spot
Block patient (stablize patch group) 28, male 16, female 12, the age 58~82 (68.3 ± 5.8) year；Optionally physical examination of healthy population 30
Example be used as healthy control group, male 16, female 14, the age 54~78 (66.7 ± 7.2) year.Cerebral infarction group patient diagnosis meets
Chinese acute ischemic cerebral apoplexy diagnosis and treatment guidelines standards in 2014, the cerebral infarction of Internal Carotid System is turned out to be through head B-sonography inspection
Extremely, and through Doppler's color ultrasound confirming merging, there are arteria carotis Vulnerable plaques.All subjects exclude active chronic inflammation and blood
Liquid systemic disease, and sign informed consent form.
Above-mentioned all patients and the corresponding serum sample of control group physical examination of healthy population are taken, and is saved in -80 DEG C.
2 carotid atherosclerosis of embodiment and corresponding healthy serum Total RNAs extraction
(1) serum sample that embodiment 1 obtains is taken out in -80 DEG C, after being slowly dissolved on ice, takes 200 μ l serum
It is transferred to centrifuge tube, 600 μ l Trizol are added are placed in vortex concussion instrument and be acutely vortexed 20s, be then stored at room temperature 5min, make core
Albumen composition sufficiently cracks, and lysate is made；
(2) 200 μ l chloroforms are added in the lysate made from step (1), acutely shakes 15s, is stored at room temperature 3min, obtain mixed
Close object A；
(3) in advance open centrifuge make its it is pre- be cooled to 4 DEG C, by mixture A made from step (2), 12000g be centrifuged
15min；
(4) it is transferred to new centrifuge tube with the supernatant fluid after pipettor aspiration step (3) centrifugation, 0.6~1 times of body is added
Long-pending isopropanol mixes, and the sample of mixing is incubated for 10min under the conditions of 15-20 DEG C, precipitates RNA；
(5) by the sample after precipitating in step (4), 12000g, abandons supernatant after 5min centrifugation, adds 75% ethyl alcohol of 1ml by 4 DEG C
Cleaning precipitating, 7500g, 4 DEG C, 5min is centrifuged to obtain RNA crude product；
(6) the RNA crude product in step (5) is dried into precipitating, adds 20 μ l DEPC water dissolution precipitating；
The present embodiment Total RNAs extraction agents useful for same is as shown in table 5:
Table 5
| Reagent | Dosage |
| Trizol | 600μl |
| Chloroform | 200μl |
| Isopropanol | Extract 0.8 times of volume of supernatant out |
| RNase-free Water | 20μl |
The total RNA of embodiment 3 is quantified and quality testing
Using the content and quality of NanDrop2000 detection serum sample RNA, to guarantee the reliability of experimental data.Always
RNA is quantitative, and the specific detection method is as follows: RNA is measured on NanDrop2000 spectrophotometer in 260nm and 280nm wavelength
Under OD value, and obtain the ratio of A260/A280, when ratio illustrates that the purity of total serum IgE is preferable between 1.8~2.0, be less than
2.0 explanations have the pollution of remaining salt ion and small molecule magazine, and when being greater than 2.0 explanations, there may be the degradations of total serum IgE.
The synthesis of 4 first chain of cDNA of embodiment
It is added in the 200 μ l EP pipes of RNase-free according to the system of table 6, with centrifuge letter from the liquid on wall after mixing
Drop, is put into PCR instrument and is reacted；
Reaction condition setting: 42-50 DEG C, 45min；70 DEG C, 15min.
Table 6
5 pcr amplification reaction of embodiment
It is added in reaction vessel according to the system of table 7, with centrifuge letter from making dripless on wall, then carry out RT- after mixing
Q PCR reaction, RT-qPCR agents useful for same are as shown in table 7:
Table 7:
Reaction condition setting: 95 DEG C of 10min；Subsequently into 95 DEG C of 15s, the circulation (totally 40 times) of 60 DEG C of 1min.
The analysis of 7 data of embodiment
The present embodiment selects to carry out relative quantification using reference gene as standard, existing reference gene 5S RNA, to target
MiRNAs is normalized, and passes through relative expression quantity of the fluorescence quantitative PCR detection target miRNAs in different samples.Table
Up to the formula of the variation of amount multiple are as follows:
△ Ct (normal serum)=Ct (normal serum miRNA)-Ct (reference gene), △ Ct (carotid atherosclerosis blood
Clearly)=Ct (carotid atherosclerosis serum miRNA)-Ct (reference gene), RQ (normal/carotid atherosclerosis)=2-△ Ct
(carotid atherosclerosis)-△ Ct (normal), wherein RQ represents relative expression quantity (Relative Quantitation, RQ),
Ct (miRNA) and Ct (reference gene) respectively represents the Ct value of target miRNAs and reference gene that fluorogenic quantitative detection arrives.It is fixed
Negative control group is set in amount experiment, and each sample carries out three multiple holes in quantitative loading and repeats.
Using 18.0 statistical software of SPSS, meet the measurement data of normal distribution withIt indicates, using single factor test side
Difference analysis carries out comparison among groups；Enumeration data is expressed as a percentage, is compared using chi-square criterion；Using Pearson correlation
For check analysis healthy control group with Vulnerable plaque with the correlation of miRNAs and blood lipid level in cerebral infarction group, P < 0.05 is poor
It is different statistically significant.
The intuitive performance for the difference analysis that miRNAs is expressed in carotid atherosclerosis serum and normal serum sample
The source that wherein error is realized by drawing the histogram containing error line is mainly the individual as present in different biological samples
Otherness is larger.
Conclusion is as follows: Vulnerable plaque is with cerebral infarction group, Vulnerable plaque group, the base for stablizing patch group and healthy control group
Plinth data and four items of blood lipid tests are horizontal.Vulnerable plaque is with cerebral infarction group, Vulnerable plaque group, stable patch group and healthy control group
The no significant difference (P > 0.05) in terms of age, gender and cerebral infarction tradition Related Risk Factors.Cerebral infarction group, no
Stablize total cholesterol level in patch group obviously to increase compared with healthy control group, statistically significant (the respectively P=of difference
0.016；P=0.003).Cerebral infarction group middle-high density lipoprotein levels are substantially reduced compared with healthy control group, and difference has statistics meaning
Adopted (P=0.001), the general indicator comparison result of 4 group objects are as shown in table 8.
Table 8:
MiR-21, miR-126, miR-155, miR-221, miR-222 are in Vulnerable plaque with cerebral infarction group, unstable plaque
Expression in block group, stable patch group and healthy control group blood plasma.Present invention discover that above 5 kinds of miRNAs are in unstable plaque
There is apparent expression difference between cerebral infarction group, Vulnerable plaque group, stable patch group and healthy control group in block,
As a result as shown in Figure 1.
MiR-21, miR-155, miR-221, miR-222 are in Vulnerable plaque with cerebral infarction group, Vulnerable plaque group, steady
The expression of patch group is determined compared with healthy control group apparent increase, and difference is statistically significant (being P=0.000).miR-
126 are substantially reduced in Vulnerable plaque with the more stable patch group of expression in cerebral infarction group, Vulnerable plaque group, difference
Statistically significant (being P=0.000), and Vulnerable plaque with miR-126 in cerebral infarction group expression less
Stablize patch group to be also substantially reduced, difference is statistically significant (P=0.007).
The correlation of miRNAs expression and four items of blood lipid tests level.Normal healthy controls are analyzed using Pearson came correlation test
Group is with Vulnerable plaque with the correlation of miRNAs and blood lipid level in cerebral infarction group.Present invention discover that the miR- in cerebral infarction group
126 expression is in obvious negative correlation (respectively P=0.037 with low-density lipoprotein, total cholesterol level；P=0.05),
As a result as shown in Figure 2.
Sequence table
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Claims (4)
1. one group for diagnosing the primer sets of carotid artery atherosclerosis plaques, which is characterized in that primer particular sequence is as follows:
2. one group for diagnosing the miRNAs markers of carotid artery atherosclerosis plaques: miR-21, miR-126, miR-155,
MiR-221 and miR-222；
Specifically, miR-21, miR-155, miR-221, miR-222 Vulnerable plaque with cerebral infarction group, Vulnerable plaque group,
The expression for stablizing patch group is increased compared with healthy control group, and miR-126 Vulnerable plaque is in cerebral infarction group, Vulnerable plaque
The more stable patch group of expression in group reduces, and Vulnerable plaque is with the expression of miR-126 in cerebral infarction group
It is also reduced compared with Vulnerable plaque group.
3. a kind of method using miRNAs diagnosis carotid artery atherosclerosis plaques, the specific steps are as follows:
(1) Carotid Artery Atherosclerotic Patients and corresponding healthy serum sample are collected；
(2) Carotid Artery Atherosclerotic Patients and corresponding healthy serum Total RNAs extraction:
1) serum sample that step (1) obtains is transferred to centrifuge tube, Trizol is added is placed in vortex concussion instrument and is acutely vortexed 10-
Then 40s is stored at room temperature 2-10min, crack nucleoprotein complex sufficiently, and lysate is made；
2) chloroform is added in the lysate made from step 1), acutely shakes 5-30s, is stored at room temperature 2-6min, obtain mixture A；
Wherein, the volume ratio of serum, Trizol and chloroform is 1:3:1；
3) open in advance centrifuge make its it is pre- be cooled to 4 DEG C, by mixture A made from step 2), 12000g is centrifuged 10-25min；
4) with pipettor aspiration step 3) supernatant fluid after centrifugation is transferred to new centrifuge tube, the different of 0.6~1 times of volume is added
Propyl alcohol mixes, and the sample of mixing is incubated for 5-20min under the conditions of 15-20 DEG C, precipitates RNA；
5) by the sample after precipitating in step 4), 12000g, abandons supernatant after 5min centrifugation, adds 0.5-2ml75% ethyl alcohol clear by 4 DEG C
Precipitating is washed, 7500g, 4 DEG C, 5min is centrifuged to obtain RNA crude product；
6) the RNA crude product in step 5) is dried into precipitating, adds solvent dissolution and precipitates and save；
Wherein, step (2) -6) in solvent be 20 μ lDEPC water；
(3) content and quality of serum sample RNA are detected；
(4) it designs, prepare specificity primer, specific primer sequence is as described in claim 1；
(5) synthesis of first chain of cDNA:
Wherein, the volume ratio of serum, Trizol and chloroform is 1:3:1；In the reaction tube of RNase-free, centrifuge is used after mixing
Letter is put into PCR instrument and is reacted from the drop on wall；
Reaction condition setting: 42-50 DEG C, 45min；70 DEG C, 15min；
(6) pcr amplification reaction:
Reagent is added in reaction vessel according to following systems, with centrifuge letter from making dripless on wall, then carry out after mixing
RT-q PCR reaction, RT-qPCR agents useful for same are as follows:
Reaction condition setting: 95 DEG C of 10min；Subsequently into 95 DEG C of 15s, the circulation of 60 DEG C of 1min, totally 40 times.
(7) data are analyzed
1) relative quantification is carried out using reference gene as standard, existing reference gene 5S RNA carries out normalizing to target miRNAs
Change processing；
2) pass through relative expression quantity of the fluorescence quantitative PCR detection target miRNAs in different samples；
Specifically, the formula of the variation of expression quantity multiple are as follows:
△ Ct (normal serum)=Ct (normal serum miRNA)-Ct (reference gene), △ Ct (carotid atherosclerosis serum)=
(neck is dynamic by Ct (carotid atherosclerosis serum miRNA)-Ct (reference gene), RQ (normal/carotid atherosclerosis)=2-△ Ct
Pulse atherosclerosis)-△ Ct (normal)；
Wherein, RQ represents relative expression quantity (Relative Quantitation, RQ)；
Ct (miRNA) and Ct (reference gene) respectively represents the Ct of target miRNAs and reference gene that fluorogenic quantitative detection arrives
Value；
3) statistical analysis is carried out to the experimental data that quantitative fluorescent PCR obtains, and passes through one-way analysis of variance target
Differential expression analysis of the miRNAs in different samples, when bilateral P value < 0.05, it is believed that the differential expression of the two is in statistics
It is upper that there is significant difference；When bilateral P value < 0.01, it is believed that the differential expression of the two statistically has extremely significant property poor
It is different.
4. miRNAs marker as claimed in claim 2 and method as claimed in claim 3 are in diagnosis carotid atherosclerosis
Application in patch.
Applications Claiming Priority (2)
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| KU-CHUNG CHEN等: "MicroRNAs in atherosclerosis", 《KAOHSIUNG JOURNAL OF MEDICAL SCIENCES》 * |
| 陈红芳等: "动脉粥样硬化性脑梗死血 miR-21、miR-126、miR-155，miR-221、miR-222 的表达水平", 《2018 年浙江省神经病学学术年会 论文汇编》 * |
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| CN112626202A (en) * | 2021-01-18 | 2021-04-09 | 上海市浦东新区周浦医院(上海健康医学院附属周浦医院) | Application of serum miRNA in auxiliary diagnosis of post-stroke cognitive impairment |
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