CN110066871A - The early diagnosis marker of hypertensive cerebral hemorrhage - Google Patents
The early diagnosis marker of hypertensive cerebral hemorrhage Download PDFInfo
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- CN110066871A CN110066871A CN201910481488.7A CN201910481488A CN110066871A CN 110066871 A CN110066871 A CN 110066871A CN 201910481488 A CN201910481488 A CN 201910481488A CN 110066871 A CN110066871 A CN 110066871A
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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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
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Abstract
The invention discloses purposes of the SETD6 gene in office hypertension cerebral hemorrhage.There are significant differences between SETD6 gene transcription level and normal person in patients with hypertensive cerebral hemorrhage blood, can distinguish patients with hypertensive cerebral hemorrhage and normal person according to difference.The invention also discloses the diagnostic products of hypertensive cerebral hemorrhage, which can realize the diagnosis of hypertensive cerebral hemorrhage by SETD6 gene in detection blood.
Description
Technical field
The invention belongs to molecular diagnosis fields, are related to a kind of molecular marker for office hypertension cerebral hemorrhage, tool
Body is related to application of the molecular marker-SETD6 gene in the product for preparing office hypertension cerebral hemorrhage in blood.
Background technique
Hypertensive cerebral hemorrhage (Hypertensive cerebral hemorrhage, HCH) system is by internal artery, vein
Or capillary rupture causes a kind of spontaneous cerebrovascular disease in brain parenchym, has anti-hypertensive properties, also known as hypertensive cerebral brain
Bleeding.Hypertensive cerebral hemorrhage is the global disease of a kind of high incidence, high disability rate and high lethality rate, is that endanger the mankind strong
Health is not only common but also serious disease.
Pathologic variation occurs for the parteriole that high blood pressure often results in brain bottom, and performance outstanding is the pipe in these parterioles
Hyaloid or fibroid degeneration drawn game focal hemorrhage, ischemic and necrosis occur on wall, weakens the intensity of vascular wall, limits to
The expansion of property, and small aneurysm can be formed.Hypertensive cerebral hemorrhage be on such pathologic basis, because it is excited,
It is excessively mental to cause blood pressure acutely to increase with manual labor or other factors, cause caused by the rupture of blood vessel in brain bleeding of lesion.
Wherein artery of cerebral hemorrhage rupture is the most common, other interior arteries after being followed successively by thalamoperforate artery, thalamus knee artery and choroid plexus
Deng.Therefore, hypertensive cerebral hemorrhage has its special predilection site, and according to large case statistics, 55% in shell core (external capsule) area,
15% in cerebral lobe subcortical white matter, 10% in thalamus, 10% pon, 10% in hemisphaerium cerebelli.And betide oblongata or in
Brain person is extremely rare.Sometimes hematoma Enlargement in Spontaneous can be in ventricular extension, but will not generally wear out cerebral cortex and cavum subarachnoidale is caused to go out
Blood.In terms of pathology, hemotoncus causes surrouding brain tissue compression, ischemic, cerebral infarction, necrosis while accompanying by serious brain edema, easily thus
Increased intracranial pressure and hernia cerebri sharply occurs.
Hypertensive cerebral hemorrhage is not equal to cerebral hemorrhage (cerebral hemorrhage, CH), and cerebral hemorrhage is also known as brain and overflows
Blood means that the angiorrhoxis in brain parenchym causes bulk bleeding to be sayed, about 80% betides cerebral hemisphere, is with coxopodite area
Main, remaining 20% betides brain stem and cerebellum.Hypertension and artery sclerosis are the principal elements of cerebral hemorrhage, can also be by congenital brain
Aneurysm, cerebrovascular malformation, brain tumor, blood disease (such as alpastic anemia, leukaemia, thrombocytopenic purpura and blood friend
Disease etc.), infection, drug (such as anticoagulant and thrombolytics etc.), outside injure poisoning etc. caused by.
At present clinically, Selected Inspection is looked into headed by head CT is unenhanced, can rapidly clear intracerebral hemorrhage position, range and hemotoncus
Amount and hemotoncus whether ventricular extension, if with subarachnoid hemorrhage etc., brain edema and cerebral infarction can also be identified.Hemotoncus
Occupation time process can be shifted by the compression of telocoele, the displacement of cerebral falx and the forfeiture of Basement geology speculate, this helps to control
The judgement of selection and the prognosis for the treatment of scheme can also identify other causes of disease, such as blood according to Hemorrhagic location and enhanced CT performance
Pipe deformity, aneurysm, tumour etc..Thought that head CT is the inspection for diagnosing cerebral hemorrhage most worthy in pervious viewpoint in 2000
Means, the detection ratio MRI high of CT, effectively.CT becomes the important detection methods of Neurology within a very long time,
Than having a wide range of application for MRI, but in 6 hours this when interim patient recall rate be not it is very high, this has been resulted in
Clinically much dissatisfied for the treatment results of cerebral infarction and patients with cerebral hemorrhage, even there is disease after the treatment in patient
The case where exacerbation.
When suspect cause the cause of disease of cerebral hemorrhage to be the factor other than hypertension when, carry out MRI inspection be it is valuable, can be with
Antidiastole cerebrovascular malformation, tumour, Giant intracranial aneurysms etc..But longer when MRI Laboratory Fee, the heavier acute case of the state of an illness
When checking, it is necessary to be guarded to the vital sign and air passage of patient, to prevent accident.In addition, the MRI of different times hemotoncus
Performance is also complex, brings difficulty to diagnosis instead sometimes.
Therefore urgently develop it is a kind of hypertensive cerebral hemorrhage early stage pass through easy detection can office hypertension brain go out
The method that blood occurs.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for hypertensive cerebral hemorrhage morning
The molecular marker of phase diagnosis.Compared to the diagnostic method of traditional hypertensive cerebral hemorrhage, height is diagnosed using gene marker
Blood pressure cerebral hemorrhage has timeliness, specificity and sensitivity, to make patient that can know disease risks, needle in disease early stage
To risk height, corresponding prevention and treatment measure is taken.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides the products of detection SETD6 gene expression in the tool for preparing office hypertension cerebral hemorrhage
Using.
Further, the product of detection SETD6 gene expression mentioned above includes: by Reverse transcription-PCR, fixed in real time
PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform SETD6 gene expression dose are measured with office hypertension
The product of property cerebral hemorrhage.
Further, the product with Reverse transcription-PCR office hypertension cerebral hemorrhage includes at least a pair of of specific amplified
The primer of SETD6 gene；The product with real-time quantitative PCR office hypertension cerebral hemorrhage includes at least a pair of of specific amplified
The primer of SETD6 gene；The product with immune detection office hypertension cerebral hemorrhage includes: and SETD6 protein-specific
In conjunction with antibody；The product in situ hybridization office hypertension cerebral hemorrhage includes: miscellaneous with the nucleic acid sequence of SETD6 gene
The probe of friendship；The product with chip office hypertension cerebral hemorrhage includes: protein chip and genetic chip；Wherein, albumen
Chip includes the antibody in conjunction with SETD6 protein-specific, and genetic chip includes the spy with the nucleic acid array hybridizing of SETD6 gene
Needle.
In specific embodiments of the present invention, the product with real-time quantitative PCR office hypertension cerebral hemorrhage is extremely
Few sequence for including the primer of a pair of of specific amplified SETD6 gene is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Preferably, the diagnostic tool includes chip, kit, test paper or high-flux sequence platform.Wherein, high pass measures
Sequence platform is a kind of special diagnostic tool, and the product of detection SETD6 gene expression can be applied to the platform and realize to SETD6
The detection of the expression of gene.It, will be to the building of the gene expression profile of a people with the development of high throughput sequencing technologies
For very easily work.Which by comparing the gene expression profile of Disease and normal population, it is easy that gene analyzed
It is abnormal related to disease.Therefore, know that the exception of SETD6 gene is related to hypertensive cerebral hemorrhage in high-flux sequence also to belong to
In the purposes of SETD6 gene, equally within protection scope of the present invention.
The present invention also provides a kind of tool of office hypertension cerebral hemorrhage, the diagnostic tool includes chip, reagent
Box, test paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for SETD6 gene transcription level
The oligonucleotide probe of SETD6 gene；The protein-chip includes solid phase carrier and the SETD6 egg for being fixed on solid phase carrier
White specific antibody；The genetic chip can be used for detecting multiple genes including SETD6 gene (for example, with high blood
Pressure property cerebral hemorrhage relevant multiple genes) expression.The protein-chip can be used for detecting including SETD6 albumen
Multiple protein (such as multiple protein relevant to hypertensive cerebral hemorrhage) expression.By by multiple with high blood
The marker of pressure property cerebral hemorrhage detects simultaneously, is greatly improved the accuracy rate of hypertensive cerebral hemorrhage diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting SETD6 gene transcription level；The protein immunization detection kit includes SETD6 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Method detects reagent needed for SETD6 gene expression dose process.Preferably, the reagent includes for SETD6 gene
Primer and/or probe.It is easy to design according to the nucleotide sequence information of SETD6 gene and can be used for detecting SETD6 gene table
Up to horizontal primer and probe.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of SETD6 gene
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe self-complementary sequence
Column are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The high-flux sequence platform includes the reagent for detecting SETD6 gene expression dose.
The test paper includes test paper carrier and the oligonucleotides that is fixed on test paper carrier, and the oligonucleotides is able to detect
The transcriptional level of SETD6 gene.
Further, the specific antibody of the SETD6 albumen includes monoclonal antibody, polyclonal antibody.The SETD6 egg
White specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with SETD6 albumen.For protein level
Antibody preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, the primer sequence for SETD6 gene is as follows: forward primer sequence
As shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
For the SETD6 gene of office hypertension cerebral hemorrhage and its source of expression product include but is not limited to blood,
Tissue fluid, urine, saliva, spinal fluid etc. can obtain the body fluid of genomic DNA.In specific embodiments of the present invention, use
In the SETD6 gene of office hypertension cerebral hemorrhage and its source of expression product be blood.
In the context of the present invention, " SETD6 gene " includes any function etc. of SETD6 gene and SETD6 gene
The polynucleotides of jljl.SETD6 gene (Chromosome 16, NC_000016.10 (58515479..58521990)) sequence
It can be inquired in international public GenBank GeneBank.
In the context of the present invention, SETD6 gene expression product includes the part of SETD6 albumen and SETD6 albumen
Peptide.The partial peptide of the SETD6 albumen contains functional domain relevant to hypertensive cerebral hemorrhage.
" SETD6 albumen " includes any functional equivalent of SETD6 albumen and SETD6 albumen.The functional equivalent
Including SETD6 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation
Body, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of SETD6 under high or low stringent condition.
It is known that, conventionally, the modification of one or more amino acid will not influence the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and wherein the change of protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by one amino acid of addition or the protein of more amino acid modification is the fusion of SETD6 albumen
Albumen.For the peptide or protein with SETD6 protein fusion, there is no limit as long as resulting fusion protein retains SETD6 egg
White biological activity.
In the context of the present invention, " office hypertension cerebral hemorrhage " both includes judging whether subject has suffered from height
Blood pressure cerebral hemorrhage also includes the risk that judges subject and whether there is with hypertensive cerebral hemorrhage.
The advantages of the present invention:
Present invention firstly discovers that SETD6 gene expression is related to hypertensive cerebral hemorrhage, by detection subject
The expression of SETD6, it can be determined that whether subject suffers from hypertensive cerebral hemorrhage or judge that subject whether there is with height
The risk of blood pressure cerebral hemorrhage, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-SETD6 genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of hypertensive cerebral hemorrhage, to reduce hypertensive cerebral hemorrhage
The death rate.
Detailed description of the invention
Fig. 1 shows the differential expression using QPCR detection SETD6 gene in patients with hypertensive cerebral hemorrhage and normal person
The histogram of situation.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens difference expression gene
1, research object
Research object clinical information is shown in Table 1.
2, in blood total serum IgE extraction
(1) homogenized (Homogenization)
Fresh blood (peripheral blood) is directly taken, 3 times of volume erythrocyte cracked liquids are added, 10 points are placed at room temperature for after mixing
Clock, 10,000rpm centrifugations 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.The leucocyte of every 100-200 μ l blood collection
1ml TRIzol is added in precipitating.
(2) it is layered (Phase Separation)
A. after TRIzol is added in sample, it is placed at room temperature for 5min, cracks sample sufficiently.
B. 200 μ l chloroforms are added in every 1ml TRIzol, and 3-5min is placed at room temperature for after shaking vigorously and mix well makes its natural split-phase.
(3) RNA precipitate (RNA Precipitation)
A.4 DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers: the organic phase of yellow, middle layer and colourless
Water phase, RNA mainly in water phase, water phase (can usually draw 550 μ l) are transferred in new pipe.
B. isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min.4 DEG C of 12,000rpm centrifugations
10min abandons supernatant, and RNA precipitate is in tube bottom.
(4) RNA rinses (RNA Wash)
75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in a.RNA precipitating, mildly vibrates centrifuge tube, it is heavy to suspend
It forms sediment.75% ethyl alcohol of 1ml is added in every 1ml TRIzol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration rapid centrifugation is carefully inhaled in abandoning with pipettor
Clearly, 1-2 minutes are placed at room temperature for and dries precipitating.
(5) RNA (Redissolving the RNA) is dissolved
50-100 μ l RNase-free water is added in precipitating, flicks tube wall, sufficiently to dissolve RNA, -70 DEG C of preservations.
3, RNA concentration and quality testing
RNA concentration: RNA Concentration Testing is carried out using Nanodrop.
RNA mass: it can be indicated by RNA integrality (RIN), it is also possible to the gel electrophoresis of plain agar sugar (deposition condition:
1.2% glue；0.5 × TBE electrophoretic buffer；150v, 15 minutes) detection integrality.
Testing result is as shown in table 2.
2 RNA concentration of table and quality measurements
4, library and sequencing process are built
1) DNase I digests: taking a certain amount of in total serum IgE sample, preparation digestion system, the thermophilic reaction in Thermomixer
Certain time utilizes DNA segment present in DNase I digestion Total RNA sample.Precipitating recycling reaction product, it is last molten
Solution is in DEPC water；
2) separation of mRNA: the total serum IgE sample of cancellation, first thermophilic is denaturalized on Thermomixer, opens its second level knot
Structure is recycled oligo (dT) Beads enrichment to go out after mRNA and is eluted using buffer to the mRNA isolated；
3) mRNA is interrupted: mRNA obtained in the previous step being placed on ice, addition interrupts reagent, in Thermomixer
Thermophilic reacts after a certain period of time, and precipitating recycling interrupts product, is dissolved in DEPC water；
4) synthesis of one chain of reverse transcription: appropriate primer being added into the mRNA after interrupting, after mixing well
Thermomixer thermophilic reacts certain time, is allowed to open secondary structure and in conjunction with primer, adds the chain prepared in advance
Synthetic reaction system synthesizes a chain cDNA by corresponding program in PCR instrument；
5) synthesis of two chain of reverse transcription: preparing two chain synthesis reaction systems, one timing of thermophilic reaction on Thermomixer
Between, two chain cDNA are synthesized, reaction product carries out purification and recovery with kit；
6) end is repaired: being prepared end and is repaired reaction system, thermophilic reacts certain time in Thermomixer, in enzyme
Under the action of, the cohesive end for the cDNA double-strand that reverse transcription obtains is repaired.End reparation product is carried out pure with kit
Change recycling, sample is finally dissolved in EB Solution；
7) 3 ' end cDNA adds " A ": it prepares and adds " A " reaction system, thermophilic reacts certain time in Thermomixer,
Under the action of enzyme, 3 ' ends for the product cDNA for repairing end are plus A base." A " product is added to be purified with kit
Recycling；
8) connection of 5 ' adapter of cDNA: preparing connector ligase reaction system, and thermophilic reacts in Thermomixer
Certain time connect connector with A base under the action of enzyme, and product carries out purification and recovery with kit；
9) connection product glue recycles: carrying out agarose gel electrophoresis to connection product, DNA fragmentation needed for cutting glue selection is big
It is small, purification and recovery is carried out with kit；
10) PCR reaction and product recycling: PCR reaction system is prepared, PCR response procedures appropriate is selected, upper step is obtained
To connection product expanded.Agarose gel electrophoresis is carried out to PCR product, DNA fragmentation size needed for cutting glue selection is used
Kit carries out purification and recovery.Recovery product is dissolved in EB solution.Labelled, library preparation is so far completed；
11) Library Quality detects: using Agilent 2100Bioanalyzer and ABI StepOnePlus Real-
TimePCR System detects Library Quality；
12) machine is sequenced on: being sequenced using Illumina Hiseq x-ten platform, PE150 strategy.Sequencing data is shown in
Shown in table 3.
3 sequencing data of table
5, bioinformatic analysis
It is as follows that sequencing data obtains later rawdata analytic process:
The shearing of 5.1 raw sequencing data quality
Due to that can include that sequence measuring joints sequence, low quality read, N rate higher sequence and length are too short in raw sequencing data
The data of sequence, this will seriously affect the quality of subsequent assembling.For guarantee subsequent bio information analysis accuracy, first to original
Beginning sequencing data is filtered, to obtain the sequencing data (clean data) of high quality.Specific step is as follows:
1) the adapter sequence in reads is removed；
2) 5 ' base of the end containing non-AGCT of removal before shearing；
3) trim the lower end reads of sequencing quality (sequencing quality value is less than Q20)；
4) ratio of the removal containing N reaches 10% reads；
5) give up adapter and quality trimming after length be less than 25bp small fragment.
Then data volume statistics is carried out to the sequence of quality trimming front and back.
Software: cutadapt, version v1.16, http://cutadapt.readthedocs.io are analyzed, parameter is-q
20-m 20.FastqStat.jar, version v1.0, custom script, parameter are default parameters.
5.2 compare analysis
By the high quality sequencing sequence obtained after Quality Control and specified reference genome alignment, research species are behaved, reference
Genome is Ensemble 92 from Ensembl database, genome version GRCh38, gene annotation information.
Software: hisat2 is analyzed, version is v 2.1.0, https: //ccb.jhu.edu/software/hisat2/
Index.shtml, parameter are-p 10--rna-strandness RF.
The assessment of 5.3mRNA expression quantity
The abundance of transcript embodies the expression of gene, and Transcript abundance is higher, then gene expression dose is higher.?
In RNA-seq analysis, the table of gene is calculated by comparing the quantity to the sequence (clean reads) with reference to genome area
Up to level.Stringtie is according to the comparison result of Hisat2 software, in conjunction with the gene annotation information of mRNA, calculate each gene/
The FPKM value of transcript in the sample, the expression quantity using the value as gene/transcript in the sample.
Analyze software: stringtie, version v1.3.3b, http://ccb.jhu.edu/software/
stringtie/.Ballgown, version v2.14.0,
http://www.bioconductor.org/packages/release/bioc/html/ballgown.html。
5.4 differential expression mRNA analysis
R packet DESeq2 compares the differential expression of each group mRNA.
DESeq2(http://www.bioconductor.org/packages/release/bioc/html/
DESeq2.html).The software is the R software package of a Recognition Different gene from RNA-Seq data, is based on bi-distribution
Model integrated Fisher is examined and likelihood ratio test carries out differential expression inspection, also provides gene standardized expression quantity to it
He tests at software.
Differential expression mRNA screening criteria are as follows: p value<0.05& | log2FoldChange |>1.Be obtained 1649 (on
1034 are adjusted, 615) a differential expression mRNA is lowered.Wherein, for comparing normal person, in patients with hypertensive cerebral hemorrhage blood
SETD6 gene expression is significantly lowered.
2 large sample of embodiment verifies difference expression gene
1, research object:
Screening criteria is with embodiment 1, patients with hypertensive cerebral hemorrhage and normal person each 30.
2, in blood total serum IgE extraction
Step is the same as embodiment 1.
3, reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgE with RT Buffer.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgE as template ribonucleic acid, following components is separately added into PCR pipe: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
4、QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of SETD6 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
SETD6 gene:
Forward primer is 5 '-AATTGTCTTCGGATGGTA-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-TTGTCAGGATATGGTTCA-3 ' (SEQ ID NO.2),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
1 PCR reaction system of table
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 10s, 52 DEG C of 40s) * 40 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries out relative quantification.
5, statistical method
Result data is indicated in a manner of mean+SD, is united using SPSS13.0 statistical software
Meter analysis, difference between the two is examined using t, it is believed that has statistical significance as P < 0.05.
6, result
The results show that having 29 patients in 30 patients with hypertensive cerebral hemorrhage compared with 30 normal person's average levels
SETD6 gene mRNA levels are significantly lowered in blood, wherein 1 excludes except statistics without significant change.Statistical result such as Fig. 1
It is shown, normal person is compared, SETD6 gene mRNA levels are significantly lowered in patients with hypertensive cerebral hemorrhage blood, and difference has system
Meter learns meaning (P < 0.05), as a result with sequencing experiment.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>the 9th 80 hospital, Chinese People's Liberation Army's joint logistics system army
<120>early diagnosis marker of hypertensive cerebral hemorrhage
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aattgtcttc ggatggta 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgtcaggat atggttca 18
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tttaactctg gtaaagtgga tat 23
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggtggaatca tattggaaca 20
Claims (10)
1. detecting application of the product of SETD6 gene expression in the tool for preparing office hypertension cerebral hemorrhage.
2. application according to claim 1, which is characterized in that the product includes: by Reverse transcription-PCR, real-time quantitative
PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform SETD6 gene expression dose are with office hypertension
The product of cerebral hemorrhage.
3. application according to claim 2, which is characterized in that described with Reverse transcription-PCR office hypertension cerebral hemorrhage
Product includes at least the primer of a pair of of specific amplified SETD6 gene；It is described with real-time quantitative PCR office hypertension cerebral hemorrhage
Product includes at least the primer of a pair of of specific amplified SETD6 gene；The production with immune detection office hypertension cerebral hemorrhage
Product include: the antibody in conjunction with SETD6 protein-specific；The product packet in situ hybridization office hypertension cerebral hemorrhage
It includes: the probe with the nucleic acid array hybridizing of SETD6 gene；The product with chip office hypertension cerebral hemorrhage includes: egg
White chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with SETD6 protein-specific, genetic chip include with
The probe of the nucleic acid array hybridizing of SETD6 gene.
4. application according to claim 3, which is characterized in that described to use the cerebral hemorrhage of real-time quantitative PCR office hypertension
The primer of a pair of of specific amplified SETD6 gene that includes at least of product as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. a kind of tool for office hypertension cerebral hemorrhage, which is characterized in that the tool can be by detection sample
The expression of SETD6 gene carrys out office hypertension cerebral hemorrhage.
6. tool according to claim 5, which is characterized in that the tool includes chip, kit, test paper or high throughput
Microarray dataset.
7. tool according to claim 6, which is characterized in that the chip includes genetic chip, protein-chip；It is described
Genetic chip includes solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, and the oligonucleotide probe includes being used for
Detect the oligonucleotide probe for SETD6 gene of SETD6 gene transcription level；The protein-chip includes solid phase carrier
And it is fixed on the specific antibody of the SETD6 albumen of solid phase carrier；The kit includes gene detecting kit and albumen
Immunity detection reagent；The gene detecting kit includes the reagent for detecting SETD6 gene transcription level；The albumen
Immunity detection reagent includes the specific antibody of SETD6 albumen；The test paper includes for detecting SETD6 gene transcription level
Reagent；The high-flux sequence platform includes the reagent for detecting SETD6 gene transcription level.
8. tool according to claim 7, which is characterized in that it is described detection SETD6 gene transcription level reagent include
For the primer and/or probe of SETD6 gene.
9. tool according to claim 8, spy are characterized in that, the primer sequence for SETD6 gene is as follows: just
To primer sequence as shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
10. the tool according to any one of claim 5-9, which is characterized in that the sample is blood.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964802A (en) * | 2019-11-11 | 2020-04-07 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Application of ApoE gene in hematoma re-expansion and prognosis evaluation in acute intracerebral hemorrhage |
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|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| CN101283106A (en) * | 2005-07-27 | 2008-10-08 | 肿瘤疗法科学股份有限公司 | Method of diagnosing small cell lung cancer |
| WO2019049144A1 (en) * | 2017-09-06 | 2019-03-14 | The National Institute for Biotechnology in the Negev Ltd. | Compositions for inhibition of the methyltransferase setd6 |
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2019
- 2019-06-04 CN CN201910481488.7A patent/CN110066871A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| CN101283106A (en) * | 2005-07-27 | 2008-10-08 | 肿瘤疗法科学股份有限公司 | Method of diagnosing small cell lung cancer |
| WO2019049144A1 (en) * | 2017-09-06 | 2019-03-14 | The National Institute for Biotechnology in the Negev Ltd. | Compositions for inhibition of the methyltransferase setd6 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964802A (en) * | 2019-11-11 | 2020-04-07 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Application of ApoE gene in hematoma re-expansion and prognosis evaluation in acute intracerebral hemorrhage |
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Application publication date: 20190730 |