CN106337089B - A kind of lncRNA for cerebral arterial thrombosis diagnosis - Google Patents

Abstract

It is LOC105376505 the invention discloses a kind of lncRNA for cerebral arterial thrombosis diagnosis, the lncRNA.Present invention firstly discovers that LOC105376505 genes are related to the occurrence and development of cerebral arterial thrombosis, express and lower in ischemic cerebral stroke patients, further enrich the research of cerebral arterial thrombosis pathogenesis.

Description

A kind of lncRNA for cerebral arterial thrombosis diagnosis

Technical field

The invention belongs to biomedicine fields, are related to a kind of lncRNA for cerebral arterial thrombosis diagnosis, specifically should LncRNA is LOC105376505.

Background technology

Cerebrovascular disease is to seriously endanger one of the three big diseases of human health together with cardiovascular disease and tumour, has height The characteristics of incidence, high disability rate and high mortality, while being also the three big cause of death of the mankind together with cardiovascular disease and tumour One of.Cerebrovascular disease is mainly to lead to the nervous system disease of local nerve afunction due to brain tissue blood circulation disorder Disease can be divided into acute cerebrovascular disease and chronic cerebrovascular disease.Acute cerebrovascular diseases are commonly referred to as cerebral apoplexy again, in the U.S., often Year, about 800,000 people suffered from cerebral apoplexy class disease, and incidence increases with the increase at age, this gives the society of its continuous astogeny Serious problem can be brought.In China, Cerebral Vascular Disease rate rises year by year, and it is lethal and disable the first of reason to have occupied population Position.And with the fast development of economic level, the continuous exacerbation of China human mortality Aging Problem and unsound life style Equal many factors so that ischemic cerebrovascular disease incidence increases year by year.Cerebral apoplexy can be divided into cerebral arterial thrombosis again (ischemic Stroke, IS) and hemorrhagic apoplexy (Hemorrhagic Stroke).Cerebral arterial thrombosis accounts for whole brain soldiers 80% or so of middle incidence, and its incidence increases with the growth at age.The generation of cerebral arterial thrombosis mainly by In the formation of cerebral embolism and local thrombus, this may be the blood vessel caused by atherosclerosis (Atherosclerosis, AS) Narrow, thromboembolism, or cause cerebral embolism with blood flow to brain from the embolus of heart；And blood caused by a variety of causes Pipe damage, vascular inflammation are also one of the major reasons.Many reasons cause brain blood to supply obstacle, and cause injured cerebral tissue Irreversibility damage so that brain tissue ischemia, anoxic lead to final necrosis, and a series of neurologic impairment is caused to patient And obstacle.

Cerebral arterial thrombosis is inherent cause and the coefficient multiple-factor complex disease of environmental factor, in addition to environmental factor Influence other than, inherent cause plays a very important role in the pathogenic process of cerebral apoplexy, existing many molecular genetics The generation that research also shows cerebral apoplexy has very high genetic predisposition.Inherent cause may be by luring Traditional Factors Hair, or as independent risk factor, eventually affect histoorgan.The diagnosis of cerebral arterial thrombosis at present depends on brain CT more How scanning, Typical AVM inspection, DSA, MRA, transcranial Doppler ultrasound inspection etc., diagnose cerebral arterial thrombosis earlier And prevent, it is the important topic studied at present.

Recently it has been found that in addition to there is microRNA, this is repaiied with robust adaptive control and epigenetic in human genome The presence of the small non-coding RNA of function is adornd, there is also ten hundreds of another non-coding RNA, that is, long-chain non-coding RNAs (long non-coding RNA, lncRNA) long-chain non-coding is the RNA molecule that a kind of transcript length is more than 200nt, they Function without coding albumen.LncRNA be initially believed to be rna plymerase ii transcription by-product, be subgenomic transcription " noise, rubbish " does not have biological function.However, it has recently been demonstrated that they can pass through DNA methylation, histone A variety of different mechanism such as modification, post-transcriptional control, RNA interference, imprinted genes, the expression water that gene is influenced in a variety of levels It is flat.Therefore, lncRNA is likely to become the new direction of cerebral arterial thrombosis research.In the prior art it has been reported that microRNA is being lacked Play an important roll in courageous and upright cerebral apoplexy occurrence and development and drug resistance, but the same lncRNA as non-coding product, life Object function and its effect during cerebral arterial thrombosis occurrence and development are unclear, require further study.

Currently, the lncRNA of the unconventionality expression found in illing tissue can relate to each system of whole body, distribution is more wide It is general, but due to lncRNA substantial amounts, it is still in the starting stage for researchs of the lncRNA in cerebral apoplexy field at present, because This molecular basis for seeking LncRNA cerebral arterial thrombosis is of great significance.

Invention content

In order to make up for the deficiencies of the prior art, an object of the present invention provides a kind of detection long-chain non-coding RNA The product of LOC105376505, to realize that the early diagnosis of cerebral arterial thrombosis provides basis.

The second object of the present invention provides a kind of diagnostic method of cerebral arterial thrombosis, by detecting lncRNA The expression of LOC105376505 come diagnose patient whether suffer from cerebral arterial thrombosis.

The third object of the present invention provides a kind of molecular marker, and the index as disease detection is applied to clinic.

The fourth object of the present invention provides a kind of and closely related gene of cerebral arterial thrombosis occurrence and development, is scarce The scientific research of courageous and upright cerebral apoplexy provides theoretical foundation.

To achieve the goals above, the present invention adopts the following technical scheme that：

The present invention provides long-chain non-coding RNA LOC105376505 in the product for preparing diagnosing ischemia cerebral apoplexy Application.Wherein, LOC105376505 refers to gene or the mRNA from the genetic transcription, and in the mankind, it is short to be located at No. 11 chromosomes On the 5th subzone of the 1st the 5th band of area of arm.

Further, the sequence of the LOC105376505 is as shown in SEQ ID NO.1.

Further, the LOC105376505 expresses downward in ischemic cerebral stroke patients.

Further, the product includes：Pass through sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies or immunoassays Method detection LOC105376505 genes expression.

Further, the nucleic acid amplification technologies are selected from PCR (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence The amplification (NASBA) of row.Wherein, PCR needs directly expand RNA reverse transcriptions at DNA (RT-PCR), TMA and NASBA before amplification Increase RNA.

In general, PCR uses denaturation, primer pair and the annealing of opposite strand and multiple cycles of primer extend, with index side Formula increases the copy number of target nucleic acid sequence；RT-PCR then is used for reverse transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in the temperature of substantial constant, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA processes Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple cycles of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple cycles of following steps：Primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand under there are dNTP α S Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting The polymerase-mediated object drawn that the ends mouth 3' carry out, which extends, to be set with replacing existing chain and generating for next round primer annealing, nicking and chain The chain changed expands so as to cause the geometry of product.

The present invention provides a kind of product of above-mentioned lncRNA LOC105376505 expressions in vitro detection sample, The product includes preparation, chip or kit." sample " include cell, tissue, internal organs, cerebrospinal fluid, body fluid (blood, Lymph etc.), digestive juice, expectoration, alveole bronchus cleaning solution, urine, excrement etc..Preferably, the sample is tissue, blood. In the specific implementation mode of the present invention, selected sample is blood.

Further, the preparation, chip or kit include the specific primer pair or probe for LOC105376505.

Further, the sequence of the specific primer pair is as shown in SEQ ID No.2 and SEQ ID No.3.

Further, the specific primer is to being used for SYBR Green, Taqman probes, molecular beacon, double cross spy The detection of needle, combined probe.

The present invention provides a kind of application of product recited above in the tool for preparing diagnosing ischemia cerebral apoplexy.

" probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts in the present invention.It removes Non- to indicate otherwise, term " probe " is often referred to match by complementary base and another polynucleotides (often referred to as " target multinuclear glycosides Acid ") combine polynucleotide probes.It is complementary to lack sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe Property target polynucleotide combine.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but not It is limited to：Solution phase, solid phase, mixed phase or in situ hybridization measuring method.

The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, Can also be RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Term " homology " refers to the degree of complementarity.There may be Homoeologies or complete homology (that is, same Property).The sequence of partial complementarity is to inhibit the nucleic acid molecules of complete complementary and " substantially homologous " target nucleic acid miscellaneous at least partly The nucleic acid molecules of friendship.Fully-complementary sequence can be by being surveyed under the conditions of low strict using hybridization with the inhibition of target sequence hybridized Fixed (Southern or Northern traces, solution hybridization etc.) is checked.Substantially homologous sequence or probe will compete simultaneously Inhibit complete homologous nucleic acid molecules under the conditions of low strict and the combination of target (that is, hybridization).This is not to say, low strict Property condition is to allow the condition of non-specific binding；It is combined into spy between low strict condition two sequences of requirement The interaction of anisotropic (that is, selectivity).Being not present for non-specific binding can be by using substantially incomplementarity (for example, low In about 30% homogeneity) the second target tested；There is no non-specific binding, probe will not be with second Incomplementarity target hybridization.

Term " hybridization " in the present invention is used to refer to the pairing of complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid Association intensity) influenced by factor such as below：The stringency of complementarity, involved condition between nucleic acid is formed Hybrid Tm and nucleic acid in G:C ratio.The individual molecule of the pairing containing complementary nucleic acid is known as " self in its structure Hybridization ".

Gene detecting kit or genetic chip can be used for detecting including LOC105376505 genes in the present invention The expression of multiple genes (for example, with the relevant multiple genes of cerebral arterial thrombosis), by multiple marks of cerebral arterial thrombosis Will object is carried out at the same time detection, is greatly improved the accuracy rate of cerebral arterial thrombosis diagnosis.

It would be recognized by those skilled in the art that the practicability of the present invention is not limited to the marker gene of the present invention The gene expression of any specific variants is quantified.As unrestricted example, marker gene can have SEQ ID NO.1 Specified cDNA sequence.In some embodiments, have and at least 85% same or analogous cDNA sequences of listed sequence Row, such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% same or analogous cDNA sequence.

Nucleic acid hybridization technique in the present invention include but not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chains as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH are for measuring with position tissue slice or entirely MRNA in organization embedding and other transcripts (for example, ncRNA).Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Term " microarray ", including but not limited to：DNA microarray (for example, cDNA microarrays and oligonucleotide microarray), Protein microarray, micro-array tissue, transfection or cell microarray, chemical compound microarray and Antibody microarray.Commonly referred to as DNA microarray for genetic chip, DNA chip or biochip is the set of microcosmic DNA points, these points are connected to the surface of solids On (for example, glass, plastics or silicon chip), formed for being carried out at the same time expression pattern analysis or expression prison to several thousands genes The array of survey.Fixed DNA fragmentation is known as probe, thousands of to can be used in single DNA microarray.Microarray can be used for passing through Compare the gene expression in disease and normal cell and identifies disease gene or transcript (for example, ncRNA).Microarray can be used Multiple technologies are manufactured, including but not limited to：Be printed onto on glass slide with apicule needle, carried out using prefabricated mask photoetching, The electrochemical method on photoetching, ink jet printing or microelectrode array is carried out using dynamic micro mirror element.

Southern and Northern traces are respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA with and the label probe of sequence of interest complementation hybridize.Detection is attached to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid fixed to film is the set of the DNA fragmentation of separation, and probe is From tissue extraction and the RNA that is marked.

Term " diagnosis " in the present invention refers to by the S＆S of disease or genetic analysis, pathological analysis, tissue The identification disease such as credit analysis.

The advantages of the present invention：

Present invention finds a kind of molecular markers of diagnosing ischemia cerebral apoplexy, by detecting marker The expression of LOC105376505 can judge early stage cerebral arterial thrombosis, to carry out early intervention, improve and suffer from The survival rate of person.

The present invention provides a kind of and closely related genes of cerebral arterial thrombosis occurrence and development, are cerebral arterial thrombosis Scientific research provide theoretical foundation.

Description of the drawings

Fig. 1 shows the difference expression gene using cDNA microarray ischemic cerebral stroke patients；

Fig. 2 shows the expression in ischemic cerebral stroke patients using QPCR detections LOC105376505.

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 is screened and the relevant gene marker of cerebral arterial thrombosis

1, sample collection

Each blood for collecting 10 normal human bloods and ischemic cerebral stroke patients, the equal informed consent of patient are above-mentioned all Agreement of the acquirement of sample by the committee of organizational ethics.

2, the preparation of RNA sample

1) 12000rpm high speed centrifugation 10min after clinical serum sample collection, are repeated 2 times, -80 DEG C of guarantors of gained serum sample It deposits；

2) 4 DEG C of defrostings of serum sample are frozen；

3) serum sample for drawing 250 μ 1 is managed to 1.5m1EP, adds 750 μ 1Trizol LS Reagent, blows and beats mixing, quiet Set 5min；

4) chlorination imitates (CHCl₃：trizol 250μ1:750 μ 1), mixing is overturned, 15min is stood；

5) 4 DEG C, 12000rpm, 15min is centrifuged；

6) supernatant liquid is carefully drawn to a new EP pipes；

7) add appropriate isopropanol (isopropanol：trizo1 500μ1：750 μ 1), mixing is overturned, 10min is stood；

8) 4 DEG C, 12000rpm centrifuges 10min；

9) upper layer centrifugate is abandoned, 75% ethyl alcohol (RNA enzyme inactivation) is added；

10) 4 DEG C, 8000rpm centrifuges 5min；

11) retain precipitation, abandon upper layer centrifugate, be stored at room temperature 5-10min；

12) appropriate DEPC water dissolutions precipitation takes appropriate RNA solution to survey concentration and observation RNA extracting situations；

13) title, concentration and date, -80 DEG C of preservations are marked.

3, reverse transcription and label

With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions at cDNA, while using Cy3 Labelling experiment group and control group respectively.

4, hybridize

Genetic chip uses Kang Cheng biology-Human lncRNA Array, is carried out by the step of chip operation instructions miscellaneous It hands over.

5, data processing

Chip Agilent scanner scannings after hybridization, resolution ratio are 5 μm, and scanner is automatically with 100% and 10%PMT Each scanning 1 time, 2 times result Agilent softwares merge automatically.Scan image data is using at Feature Extraction Reason analysis, obtained initial data application Bioconductor program bags carry out follow-up data processing.Last Ratio values are experiment Group and control group.Differential gene screening criteria：Ratio >=4 are up-regulation gene, and ratio≤0.25 is down-regulated gene.

6, result

The results are shown in Figure 1, compared with healthy human blood, expression of the LOC105376505 in ischemic cerebral stroke patients Level is substantially less than the expression of Healthy People.

The differential expression of 2 QPCR sequence verification LOC105376505 genes of embodiment

1, large sample QPCR verifications are carried out to LOC105376505 gene differential expressions.It is received according to the sample in embodiment 1 Mode set selects each 100 of the blood sample sample of normal person and ischemic cerebral stroke patients.

2, RNA extraction steps are the same as embodiment 1.

3, reverse transcription：

(1) reverse transcription reaction：

Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgEs as template ribonucleic acid, is separately added into PCR pipe following Component：DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV, Template ribonucleic acid.42 DEG C are incubated 1h, 72 DEG C of 10min, of short duration centrifugation.

(2) design of primers：

The primer sequence of LOC105376505 genes is：

Forward primer：5’-AACTGAAGAACCACAAGAGAAG-3’(SEQ ID NO.2)

Reverse primer：5’-TTGCTGTCCAGGAGAAGG-3’(SEQ ID NO.3)

The primer sequence of house-keeping gene GAPDH is：

Forward primer：5’-CCGGGAAACTGTGGCGTGATGG-3’(SEQ ID NO.4)

Reverse primer：5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’(SEQ ID NO.5)

(3) QPCR amplifications are examined：

Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect Demonstrate,prove the reliability of result.

Prepare following reaction system：12.5 μ l of SYBR Green PCRs system, forward and reverse primer (5 μM) are each 1 μ l, template cDNA 2.0 μ l, 8.5 μ l of no enzyme water.Operations are carried out on ice.

Amplification program is：95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 40 cycles.

Using SYBR Green as fluorescent marker, it is anti-that PCR is carried out on Light Cycler fluorescence real-time quantitative PCR instrument It answers, determines that purpose band, Δ Δ CT methods carry out relative quantification by melt curve analysis analysis and electrophoresis.

3, statistical method

Experiment is all completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical softwares come for statistical analysis, difference between the two is examined using t, it is believed that works as P<Have when 0.05 It is statistically significant.

4, result

The results are shown in Figure 2, compared with Healthy People, under LOC105376505 genes are expressed in ischemic cerebral stroke patients It adjusts, difference has statistical significance (P<0.05), consistent with RNA-sep results.

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present 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 domain of the claims in the present invention.

SEQUENCE LISTING

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Claims (9)

1. detection reagent the answering in the product for preparing diagnosing ischemia cerebral apoplexy of long-chain non-coding RNA LOC105376505 With.

2. application according to claim 1, which is characterized in that the sequence of the LOC105376505 such as SEQ ID NO.1 It is shown.

3. application according to claim 1 or 2, which is characterized in that the LOC105376505 suffers from cerebral arterial thrombosis It expresses and lowers in person.

4. application according to claim 1 or 2, which is characterized in that the product includes：Sequencing technologies, nucleic acid hybridize skill Reagent used in art, nucleic acid amplification technologies or the method for immunoassays.

5. application according to claim 4, which is characterized in that the nucleic acid amplification technologies be selected from PCR, Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification.

6. a kind of production of the lncRNA LOC105376505 expressions as claimed in claim 1 or 2 in vitro detection sample Product, it is characterised in that the product includes preparation, chip or kit.

7. product according to claim 6, which is characterized in that the preparation, chip or kit include being directed to The specific primer pair or probe of LOC105376505, wherein the sequence of the specific primer pair such as SEQ ID No.2 and SEQ Shown in ID No.3.

8. product according to claim 7, which is characterized in that the specific primer to be suitable for SYBR Green, The detection of Taqman probes, molecular beacon, double cross probe, combined probe technology.

9. application of the claim 6-8 any one of them product in the tool for preparing diagnosing ischemia cerebral apoplexy.