CN107142316B - Diagnosis of glaucoma molecular marked compound lncRNAsT267384, kit and application - Google Patents
Diagnosis of glaucoma molecular marked compound lncRNAsT267384, kit and application Download PDFInfo
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- CN107142316B CN107142316B CN201710423197.3A CN201710423197A CN107142316B CN 107142316 B CN107142316 B CN 107142316B CN 201710423197 A CN201710423197 A CN 201710423197A CN 107142316 B CN107142316 B CN 107142316B
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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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses molecular marked compound lncRNAsT267384, kit and the applications for diagnosis of glaucoma.It is found by the applicant that relative to control group crowd, lncRNAs:T267384 equal up-regulated expressions in the aqueous humor and serum of glaucoma patient.Prompt T267384 is the high expression lncRNAs in glaucoma, contributes to the biomarker of diagnosis of glaucoma.The present invention provides strong molecular biology mechanism for diagnosis of glaucoma, has far-reaching clinical meaning and generalization.
Description
Technical field:
The present invention relates to molecular diagnostic techniques fields, and in particular to a kind of long-chain non-coding contributing to diagnosis of glaucoma
RNA markers, kit and application.
Background technology:
Eyes are the highly important sense organs of human body, can receive external light stimulus, and light impulsion is transmitted to greatly
Mesencephalic centre and cause vision.Leonardo da Vinci once said：" the eyes are the windows of the mind, and by eyes, people are able to embrace and appreciate the world
It is unlimited beautiful, soul is just able to live in peace in vivo ".Information age, in the information that people is obtained from the external world by sense organ, greatly
About 90% is completed by eyes.The data of the World Health Organization shows that ophthalmology disease has become after tumour, angiocardiopathy
The disease of the harm of third position and influence people's life quality later.In all ophthalmology diseases, glaucoma is then that first place can not
The reason of blinding eye disease reversed, influence visual quality is to cause to lose by threatening and damaging optic nerve and its access
It is bright, to seriously threaten the Vision Health of the mankind, loss difficult to the appraisal is caused to personal, family and society.
Main harm caused by glaucoma is to influence visual performance, even in developed country, also only 50% or so
Glaucoma patient can obtain timely diagnosing and treating, moreover, the pathogenic factors of glaucoma, genetics law etc. are unknown
, therefore we will scientifically grasp its regularity of occurrence and development, to carry out early diagnosis and early treatment, glaucoma be avoided to suffer from
The blindness of person.Currently, the diagnosis of glaucoma is mainly by the inspection in terms of medical history, morphology and function assessment, as tonometry,
Ultrasound biomicroscopy UBM, eye-ground photography, optical coherence tomography and perimetry etc..Although these inspections can diagnose green light
Eye, but studies have shown that the glaucoma diagnosed by morphology and function assessment means, the damage of patient visual's function already exceed
50%.And the relevant biochemical analysis of glaucoma, serological screening and examination criteria be still in opposite space state, therefore find one
The highly sensitive marker with high specific of kind to diagnosis of glaucoma and monitors particularly important.
Genome plan studies have shown that composition human genome 3,000,000,000 base-pairs in, only 1.5% nucleotide
Sequence is used for protein coding, remaining 98.5% genome is non-protein coded sequence.These sequences be once considered as into
Change " determined garbage sequence " accumulated in the process and do not paid close attention to, in human genome DNA's element encyclopedia of subsequent start-up
(encyclopedia of DNA elements, ENCODE) in the works, research shows that 75% gene order can be transcribed
At RNA, wherein nearly 74% transcription product is non-coding RNA (non-coding RNAs, ncRNAs).In ncRNA, sequence
Length be more than 200 bases transcript be long-chain non-coding RNAs (long noncoding RNA, lncRNAs), they by
It is concerned in rich and mechanism of action the diversity of function.LncRNAs can transcription, transcription after and translation it is multiple
The expression that protein coding gene is adjusted in level, to widely participate in the important life including cell differentiation and body development
Life process, unconventionality expression are also closely related with the generation of a variety of major diseases of the mankind.
LncRNA has well-conserved and tissue specificity, very abundant in brain content.LncRNA not only takes part in
The growth and development and perfect in shape and function of nervous system make nervous system be carried out according to regular hour sequence and in certain space
Growth and development, and participate in executing the function of nervous system.LncRNA participates in the development of nervous system by number of mechanisms
And during function executes, including as cis-acting elements and trans-acting factor participation Genomic Imprinting, chromatin remodeling, cell week
The processes such as period regulation, montage regulation and control, mRNA degradations and translational control.Therefore, the composition and expression of detection lncRNAs are utilized
Variation come reflect nervous system physiology and pathological state be feasible means.
It is tolerant that aqueous humor belongs to intraocular, is generated by ciliary body, eventually enters into blood by pupil, trabecular network etc., and in dynamic
Among state cycle, ingredient constitutes closely related with eyeball local physiological and pathology environment.Excretion body is most important as body
Intermediary's substance for mutually exchanging of iuntercellular, contain lncRNAs, mRNAs, protein and lipid etc..Include in excretion body
Substance is due to special protection mechanism energy stable for extended periods of time state.Therefore, the lncRNAs equimoleculars in aqueous humor can be with excretion body
Form transmits intercellular mutual exchange of information, and can enter blood with the dynamic circulation of aqueous humor.Therefore glaucoma is relevant
LncRNAs researchs have detects the potential provided fundamental basis for glaucoma associated biomarkers.
Invention content
An object of the present invention is to provide a kind of long-chain non-coding RNA for glaucoma early diagnosis, to glaucoma
Morbidity detection it is significant.
Molecular marked compound lncRNAsT267384, sequence such as SEQ ID NO for glaucoma early diagnosis:Shown in 1.
The second object of the present invention is to provide the application of above-mentioned molecular marked compound lncRNAsT267384.Detection
Application of the product of lncRNAsT267384 expressions in preparing glaucoma early diagnosis tool.
The product of the detection lncRNAsT267384 expressions includes：It is detected by real-time fluorescence quantitative PCR
The preparation of lncRNAsT267384 expressions.
The preparation that lncRNAsT267384 expressions are detected with real-time fluorescence quantitative PCR includes specific amplification
The primer of lncRNAsT267384.
The specific amplification lncRNAsT267384 of lncRNAsT267384 expressions is detected with real-time fluorescence quantitative PCR
Primer sequence be：
F:5'- TCTGGGCATTGAAGAGTGAAG-3 ',
R:5'-AGAGGGATGGGTACAAATAAGC-3’。
The third object of the present invention is to provide a kind of kit for glaucoma early diagnosis, including detection
The reagent of lncRNAsT267384 expressions.Specifically include that lncRNAsT267384 is detected by real-time fluorescence quantitative PCR
The reagent of expression.
The reagent that lncRNAsT267384 expressions are detected by real-time fluorescence quantitative PCR includes by real-time
The primer of quantitative fluorescent PCR specific amplification lncRNAsT267384.
The reagent that lncRNAsT267384 expressions are detected by real-time fluorescence quantitative PCR passes through comprising a pair
The primer of real-time fluorescence quantitative PCR specific amplification lncRNAsT267384, primer sequence are：
F:5'- TCTGGGCATTGAAGAGTGAAG-3 ',
R:5'-AGAGGGATGGGTACAAATAAGC-3’。
We exist with the verification of qRT-PCR detection methods lncRNAs-T267384, ENST00000607393 and T342877
Expression in aqueous humor, iris tissue and serum and the difference for analyzing its expression quantity in two class aqueous humors.In being organized at three kinds,
There is aqueous humor highest diagnostic significance, sensibility/specificity that three kinds of lncRNAs diagnose glaucoma in aqueous humor to be all higher than
90%.The possible cause of this phenomenon is the presence due to blood aqueous barrier, significantly limits haematogenous immunocyte and comes from
The signaling molecule at other positions of whole body enters aqueous humor.Therefore compared with serum for, aqueous humor more can specificity representative eye physiology
And pathological state.Therefore, aqueous humor may have the diagnosis of glaucoma of bigger to be worth compared with serum in research from now on.
For the application, it is found by the applicant that relative to control group crowd, lncRNAs:T267384 is in glaucoma patient
Aqueous humor and serum in equal up-regulated expression.Prompt T267384 is the high expression lncRNAs in glaucoma, contributes to glaucoma
The biomarker of diagnosis.The present invention provides strong molecular biology mechanism for diagnosis of glaucoma, has far-reaching face
Bed meaning and generalization.
Description of the drawings
Fig. 1 is lncRNAs and mRNAs express spectras in glaucoma patient aqueous humor；
A：LncRNAs expression pattern analysis dendrogram in glaucoma patient aqueous humor；B：MRNAs is expressed in glaucoma patient aqueous humor
Spectrum analysis dendrogram.
Fig. 2 is the lncRNAs and mRNAs of differential expression in glaucoma patient aqueous humor；
A：For age-related cataract, the lncRNAs of 2 times of differential expressions in glaucoma patient aqueous humor；B：Compared with year
For age related cataract, the mRNAs of 2 times of differential expressions in glaucoma patient aqueous humor.
Fig. 3 is the CNC analysis charts of lncRNAs and mRNAs expression in glaucoma patient aqueous humor；
Red point represents lncRNAs, and blue dot represents mRNAs, and solid line represents positive correlation, and dotted line represents negative correlation.
Fig. 4 is expression quantity of the lncRNAs in glaucoma and patients with senile cataract aqueous humor；
A-C：T267384, ENST00000607393 and T342877 are in glaucoma and patients with senile cataract room
The scatter plot expressed in water；D-F：T267384, ENST00000607393 and T342877 are interior in vain in glaucoma and age related
The case figure expressed in barrier patient's aqueous humor.
Fig. 5 is expression quantity of the lncRNAs in glaucoma patient compares crowd's iris tissue；
A-C：T267384, ENST00000607393 and T342877 are in glaucoma patient and control crowd's iris tissue
The scatter plot of expression；D-F：T267384, ENST00000607393 and T342877 compare crowd's iris tissue in glaucoma patient
The case figure of middle expression.
Fig. 6 is expression quantity of the lncRNAs in glaucoma patient and control crowd's serum；
A-C：T267384, ENST00000607393 and T342877 are expressed in glaucoma patient and control crowd's serum
Scatter plot；D-F：What T267384, ENST00000607393 and T342877 were expressed in glaucoma patient compares crowd's serum
Case figure.
Fig. 7 is the ROC curve for diagnosing glaucoma in different tissues using lncRNAs；
A：The ROC curve of glaucoma, area under the curve 0.998 are diagnosed in aqueous humor using T267384；B：It utilizes
ENST00000607393 diagnoses the ROC curve of glaucoma, area under the curve 0.998 in aqueous humor；C：Existed using T342877
The ROC curve of glaucoma, area under the curve 0.983 are diagnosed in aqueous humor；D：Using ENST00000607393 in iris tissue
Diagnose the ROC curve of glaucoma, area under the curve 0.793；E：The ROC for diagnosing glaucoma in serum using T267384 is bent
Line, area under the curve 0.620；F：The ROC curve of glaucoma is diagnosed in serum using ENST00000607393, under curve
Area is 0.638.
Specific implementation mode
It is intended to further illustrate the present invention with reference to embodiments, is not intended to limit the present invention.
Embodiment 1：The express spectra of lncRNAs and mRNAs in glaucoma patient aqueous humor：
1.1 materials and reagent
1.1.1 key instrument
Common centrifuge and refrigerated centrifuge are purchased from Eppendorf companies；Supercentrifuge is purchased from Beckman companies；
Real-Time PCR instruments are purchased from ABI companies；Pipettor and electrical pipette rifle are purchased from Eppendorf companies；Q5000 is purchased from
Quawell Technology companies；Ice machine is purchased from Sanyan companies.
1.1.2 material and reagent
The centrifuge tube and PCR pipe of different model are purchased from Axygen companies；DEPC water is purchased from prosperity company of ancient cooking vessel state；Trizol
Purchased from Invitrogen companies；TargetAmpTM1-Round aRNA Amplification kits are public purchased from epicentre
Department；Transcriptor First Strand cDNA Synthesis kits are purchased from Roche companies；Quick Amp
Labeling Kit, One-Color kits are purchased from Agilent Technologies companies；People lncRNAs microarray
Chip is purchased from Arraystar companies.
1.1.3 reagent configures
Various immunoblotting agents useful for same according to《Molecular cloning (third edition)》The method of middle offer configures.
1.2 method
1.2.1 aqueous humor samples prepare
The research is examined by Ethics Committee of Xiangye No. 2 Hospital of Central South University.It is preoperative to know with the signature of all research objects
Feelings letter of consent extracts undiluted room using the disposable sterilized injector of 1ml before formal operating procedure starts in corneal limbus
This 0.1ml of water gauge is injected immediately in autoclaved 0.5mlEP pipes, be placed in -80 DEG C of low temperature refrigerators be kept in dark place it is spare.It is all
Selected patient is to be hospitalized to need to carry out the glaucoma patient and patients with senile cataract of operative treatment in our hospital.It is included in mark
It is accurate：1. primary open-angle glaucoma：A. Yan Ya >21mmHgB. glaucomatous disks damage and/RNFL defects C. is typically green
Light eye defect of visual field D. anterior chamber angles open.With above four or with A therein, D, B or C person.2. normal tension is green
Light eye：With the optic disk change similar to POAG, RNFL and visual field damage, tonometry for 24 hours≤21mmHg, room angle opens.
3. primary angle-closure glaucoma：Optic disk with glaucoma changes, RNFL and visual field damage, Yan Ya >21mmHg, anterior chamber angle become
Narrow or 4. age-related cataracts of closing (control group)：Crystalline lens is mixed in property under cortex and/or caryogram is muddy and/or rear capsule
It is turbid.Exclusion criteria：1. occur together systemic disease such as hypertension and diabetes etc..2. secondary glaucoma 3. may influence the visual field,
Optic nerve or the ophthalmology or neuropathy of colour vision.4. reliability standard is detected in the visual field：Fixation Loss Rate, false positive rate and/or
Jia Yinxingshuai >25%.5. age-related cataract：It excludes concurrency, traumatic, congenital cataract and excludes with other
Ophthalmic disease person excludes overripe stage case.
1.2.2 RNA is extracted
It takes aqueous humor 0.1ml that 0.5mlTrizol reagents are added, 0.1 milliliter of chloroform acutely is added after concussion.After and through centrifugation,
10 microlitres of DPEC water are dissolved in after precipitation and the cleaning of 75% ethyl alcohol, through each pipe RNA concentration of Q5000 apparatus measures.
1.2.3RNA amplification
Use TargetAmpTM1-Round aRNA Amplification Kit 103 (epicentre) press raw manufacturer and say
Step expands RNA shown in bright book.Substantially steps are as follows：It is single at single-stranded cDNA according to the synthesis of RNA samples first:RNA is miscellaneous
Product is handed over, it is small fragment sequence then to use RNase H enzymes to digest above-mentioned hybrid product, these small fragment sequences can assist double
The synthesis of chain cDNA.Antisense RNA is finally synthesized by double-strand cDNA transcriptions.
1.2.4 cDNA synthesis and label
Raw manufacturers instruction is pressed using Transcriptor First Strand cDNA Synthesis Kit (Roche)
Shown step synthesizes cDNA.It is right using Quick Amp Labeling Kit, One-Color (Agilent Technologies)
The cDNA of synthesis is marked.
1.2.5 labeling effciency quality testing
1.5 microlitres of marked cDNA samples are taken, fluorescent label efficiency is detected using NanoDrop ND-1000.
1.2.6 chip hybridization
At the standard conditions by the probe marked and superchip (Human lncRNA microarray V4.0,
Arraystar companies) hybridized.The chip detects 40173 kinds of lncRNAs and 20730 kinds of mRNAs expressions altogether.
1.2.7 Image Acquisition and data analysis
The fluorescence intensity of chip is scanned using GenePix 4000B chip scanners, and experimental result is converted into number
Type data preserve.P values <0.05 is statistically significant for difference.
1.3 result
1.3.1 in glaucoma patient aqueous humor lncRNAs and mRNAs express spectra
In 10 parts of glaucoma patient aqueous humor samples average detected to 20653 ± 569.9 kinds of lncRNAs and 11265 ±
268.3 kinds of mRNAs.Wherein, the lncRNAs detected in 10 parts of glaucoma patient aqueous humor samples is 11728 kinds, is detected
MRNAs be 6686 kinds.
1.3.2 in glaucoma patient aqueous humor differential expression lncRNAs and mRNAs
For age-related cataract, in glaucoma patient aqueous humor the lncRNAs of 2 times of up-regulated expressions be 4372 kinds, 2
The lncRNAs for lowering expression again is 2602 kinds.For age-related cataract, 2 times of upper mileometer adjustments in glaucoma patient aqueous humor
The mRNAs reached is 2783 kinds, and 2 times of mRNAs for lowering expression are 1617 kinds.
1.4 result
Since easily taking property, individual specificity and opposite other organs by body are influenced smaller feature for it, aqueous humor is a kind of
Eye disease biomarker correlative study is worth larger research object.Also, although there are many research reports at present
The relevant epidemiology of glaucoma and science of heredity risk factor, but it is relatively fewer as the correlative study of research object using aqueous humor.Cause
This, it is believed that aqueous humor has the potential for being applied to glaucoma correlated inheritance research from now on.Due to the limitation of sampling, humanized
Aqueous humor samples small volume.To overcome the problems, such as this, we increase RNA amplification step before carrying out chip hybridization, so to ensure
Subsequent step is smoothed out.By using lncRNAs chip microarray analysis methods, 11728 kinds of lncRNAs and 6686 kinds
MRNAs detects in 10 parts of Aqueous Humor of Glaucomas samples, in this age-related cataract patient aqueous humor lncRNAs and
MRNAs express spectras have significant difference, therefore lncRNAs and mRNAs express spectras show individual specificity and disease in aqueous humor
Specificity.As far as we know, this is first Aqueous Humor of Glaucomas correlation lncRNAs and mRNAs express spectra research.
Embodiment 2：The correlation of specific lncRNAs and mRNAs in clear aqueous humor is analyzed by CNC
Further to inquire into the possibility function of the lncRNAs expressed in glaucoma patient aqueous humor, we use CNC
The clear mRNAs expression with glaucoma related gene of (coding-noncoding gene co-expression) analysis has
The lncRNAs of significant correlation.CNS analyses are that one kind co-expressing data by lncRNA and mRNA, and lncRNA and mRNA is joined
The analysis method that system gets up.It is analyzed by CNC it can be found that with mRNAs of some lncRNA with identical expression pattern, passes through this
The function of a little mRNA, can connect lncRNA and signal specific access or disease condition, consequently facilitating prediction lncRNA
Function, and disclose its mechanism of action.
2.1 method
It selects differential expression in aqueous humor and there is the mRNAs of correlation with glaucoma occurrence and development, these mRNAs are existed
Expression data in different glaucoma patient's aqueous humor are averaged；Data and green light after asking the mRNAs picked out to standardize
Pearson correlation coefficient (Pearson Correlation in eye patient's aqueous humor between the lncRNAs related datas of differential expression
Coefficient, PCC) and false discovery rate (False Discovery Rate, FDR)；Pick out PCC >=0.90 and FDR
≤ 0.05 record；Using relative recording, drawn using Cytoscape2.8.3 tools.
2.2 result
For age-related cataract, in glaucoma patient aqueous humor differential expression and with glaucoma occurrence and development have
It is following ten kinds to have the mRNAs of correlation：Bone morphogenetic protein 2 (bone morphogenetic protein 2, BMP2),
Endyma related gene 1 (ependymin related 1, EPDR1), transforminggrowthfactor-β1 (transforming growth
Factor beta 1, TGFB1), plug albumen E3 (forkhead box E3, FOXE3), secretagogue receptor
(growth hormone secretagogue receptor, GHSR), plug PROTEIN C 1 (forkhead box C1,
FOXC1), cross-film and coiled-coil domain 1 (transmembrane and coiled-coil domains 1, TMCO1),
PH structural domains A7 (pleckstrin homology domain containing A7, PLEKHA7), optic nerve albumen
(optineurin, OPTN) and integrin subunit β 5 (integrin subunit beta 5, ITGB5).In glaucoma patient room
In water, being more than 0.9 and lncRNAs of the false discovery rate less than 0.05 with the Pearson correlation coefficient of these mRNAs expression has
10 kinds.
Embodiment 3
We verify 10 kinds in embodiment 2 using qRT-PCR in glaucoma and age correlation cataract patient aqueous humor
In lncRNAs, differential expression measurers of 7 kinds of lncRNAs in two class aqueous humors has significant difference, and 3 kinds of lncRNAs are in two class rooms
Differential expression amount no difference of science of statistics in water.Then choose the most significant 3 kinds of T267384 of differential expression amount,
ENST00000607393 and T342877, is further verified.
Embodiment 4
We with qRT-PCR detection methods verification embodiment 3 in the most significant 3 kinds of lncRNAs molecules of difference aqueous humor,
Expression in iris tissue and serum, and analyze the difference of its expression quantity in glaucoma patient and normal person.
4.1 materials and reagent
4.1.1 key instrument
Common centrifuge and refrigerated centrifuge are purchased from Eppendorf companies；Supercentrifuge is purchased from Beckman companies；
Real-Time PCR instruments are purchased from ABI companies；Pipettor and electrical pipette rifle are purchased from Eppendorf companies；Q5000 is purchased from
Quawell Technology companies；Ice machine is purchased from Sanyan companies.
4.1.2 material and reagent
The centrifuge tube and PCR pipe of different model are purchased from Axygen companies；DEPC water is purchased from prosperity company of ancient cooking vessel state；Trizol
Purchased from Invitrogen companies；Reverse Transcriptase kit and fluorescence quantitative kit are purchased from Roche companies.
4.1.3 reagent configures
Various immunoblotting agents useful for same according to《Molecular cloning (third edition)》The method of middle offer configures.
4.2 method
4.2.1 aqueous humor, serum and iris sample prepare
Aqueous humor samples are prepared by step described in 1.2.1.Serum sample is derived from glaucoma patient group and healthy control group crowd,
Wherein glaucoma patient be included in and exclusion criteria is with described in 1.2.1, control group serum be derived from glaucoma groups patient age with
The matched healthy population of gender.Iris sample is derived from glaucoma patient group and control group, and wherein glaucoma patient being included in and arranging
Except standard is with described in 1.2.1, control group iris sample is derived from and glaucoma patient age and refined two hospital in the matched Hunan of gender angle
Film donates the iris tissue of volunteer.
4.2.2 RNA is extracted
0.5 milliliter of Trizol reagent is added in 0.1 milliliter of aqueous humor or iris tissue, and 0.1 milliliter of chloroform acutely is added after concussion.
20 microlitres of DPEC water are dissolved in after centrifugation, precipitation and the cleaning of 75% ethyl alcohol after and, through each hole RNA concentration of Q5000 apparatus measures.Outside
All venous blood is put into after standing 30 minutes in centrifuge, is centrifuged 10 minutes under conditions of 3200 revs/min, is used the shifting of sterilizing
Liquid pipe takes upper serum, so extracts RNA via above-mentioned same step.
4.2.3 qRT-PCR
Reverse transcriptase is relied on first by RNA reverse transcriptions into cDNA according to raw manufacturers instruction, is then expanded with PCR method
CDNA detects the product amount of quantitative amplification by measuring fluorescence intensity signals in real time.
4.2.2 data analysis
Data processing is carried out using SPSS19.0 editions statistics softwares.Measurement data is with median ± interquartile range table
Show, glaucoma/cataract represents two groups of median ratios, compares and carries out Mann-whitney U inspections.P values <0.05 is difference
It is statistically significant.
4.3 result
Exist further to expand the expression of aqueous humor samples amount verification correlation lncRNAs and probing into specific lncRNAs
Expression quantity in glaucoma patient serum and iris, we detect 60 aqueous humor samples (30 glaucoma patients using qRT-PCR
Aqueous humor samples, 30 ages and the matched patients with senile cataract aqueous humor sample of gender), 50 iris tissues (40
Glaucoma patient iris tissue sample, the iris tissue at 10 ages and the matched cornea donations volunteer of gender) and 158 blood
In final proof sheet (103 glaucoma patient serum samples, 55 ages and the matched healthy population serum sample of gender)
T267384, ENST00000607393 and T342877 expression.The result shows that in two class aqueous humor samples, three kinds
The expression quantity of lncRNAs has significant difference, and expression quantity is followed successively by white in age related in glaucoma patient aqueous humor
4.4036,2.1467 and 2.9692 times of expression quantity in cataract or glaucoma patient's aqueous humor；The ENST00000607393 in two class iris tissues
Expression quantity have significant difference, in glaucoma patient iris tissue expression quantity be expressed in control group iris tissue
3.3436 times of amount, and the expression quantity no difference of science of statistics of T267384 and T342877；In two class serum samples, T267384
And the expression quantity of ENST00000607393 has significant difference, expression quantity is followed successively by right in glaucoma patient serum
According to 1.2878 and 1.5301 times of expression quantity in group serum, and expression quantity no difference of science of statistics (table 1,2, the 3) (figure of T342877
4,5,6).The validity of glaucoma is diagnosed to further appreciate that these three lncRNAs in not homologue, we utilize gained
Data draw Receiver Operating Characteristics (receiver operating characteristic, ROC) curve (Fig. 7).It was found that
T267384, ENST00000607393 and T342877 diagnose Line Integral under the ROC curve of glaucoma in aqueous humor samples
0.998,0.998 and 0.983.ENST00000607393 diagnoses area under the ROC curve of glaucoma in iris tissue
0.793；T267384 and ENST00000607393 diagnoses Line Integral under the ROC curve of glaucoma in serum sample
0.620 and 0.638.When the diagnostic value of T267384, ENST00000607393 and T342877 in aqueous humor is set as 1.5437,
When 1.1485 and 2.1052, sensibility/specificity of diagnosis is followed successively by 0.967/0.967,0.967/0.967 and 0.967/
0.900；When diagnostic values of the ENST00000607393 in iris tissue is set as 0.2470, sensibility/specificity of diagnosis
For 0.700/0.700；When the diagnostic value of T267384 and ENST00000607393 in serum is set as 0.7191 and 0.8376,
Its sensibility/specificity diagnosed is followed successively by 0.612/0.600 and 0.680/0.600.
Expression quantity of the 13 kinds of lncRNAs of table in aqueous humor
Expression quantity of the 23 kinds of lncRNAs of table in iris tissue
Expression quantity of the 33 kinds of lncRNAs of table in serum
The sensibility and specificity that 43 kinds of lncRNAs of table are diagnosed in different tissues
SEQUENCE LISTING
<110>Central South University
<120>Diagnosis of glaucoma molecular marked compound lncRNAsT267384, kit and application
<130>Nothing
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 6891
<212> DNA
<213> lncRNA：The sequence of T267384
<400> 1
tatttcatct tcgcattatt ttcaatactg aaggtaaatt tagatctctc tttttttctt 60
agtctggcta aaagcttgtc aattttgctt aacttttcaa aaaaccaact ttttgtttca 120
ttgatttttg tattgtttac ttcattttaa tttcatttat ttctgttctg atctttatta 180
tttcttctac taattttgga tttggtttgc tcttgttttc ctaattcttt aagatgcatc 240
attagattgt ttatttaatg taggcactat aagtttccct cttttttgat gtaggcacta 300
taacgataag cttccctctt agtactgctt ttgctgtatc ccatagattt tggtatgttg 360
tgctcccatt agcatttgtt ccaagaaatt tttaaatttc ttattaaaat gatccatcct 420
tgagaatgat ccatatgctg aggaaaagca tgtgtattcc acagccattg gatgaaatgt 480
tccgtaatta tctactggat ccatttggtc tattgtgcag attaagtctg atgtttcttt 540
attgattttt ctgcctggaa gatccatcca gtgctgaaag tggggtgtcg aagtctccag 600
ctattattgt attggggcct aactctctct ttagctcttt tttttgtttt gttttgtttt 660
gagatggagc ctggctctgt tgcccagggt ggagtgcagt ggtatgatct tggctcactg 720
caacctccgc ctcccaggtt caagctattc tcctgcctca gcctcccaag tagctgggat 780
tacagacatg tgccaccata cccggctaat tttgtatttt tagtagagat agggtttctc 840
catcttggtc aagctggtct cgaactccca acctcaggtg atccacctgc cttggcctcc 900
ccaaagtgct gggattatag gcatgagcca ctgcgcctgg ccttttttat ttttttaagg 960
aggggggggg tctcactaca ttgtctaggc tagactcaaa ctcctgggct caagtgatcc 1020
ttctgcctcg acctcctgaa atgctgtgat tacaggcatg agccaccatg actggccctg 1080
actgtgtatt ttcaaatagc ctgtcttcag gctcactaat tctttcttct gcttgatcaa 1140
ttctgctact gaaagcctct aatgcattgt tcagtatgcc aattgcattt ttagctctgg 1200
aatctcagtt tgattctttt tattttaatc tttttgttaa atgtatctga tataattttg 1260
aattccttct ctgtgttatc ttgaatttct tgagtttcct caacacagct attttgaatt 1320
ctctgcctga aaggtcacac atctctgttt ctccaggatt ggtccctggt ggctcattta 1380
gttcatttgg gaggtcatgt tttcctggat ggtgttgatg ttagtaaatg ttcttcagtg 1440
tctgggcatt gaagagtgaa gtatttattg tagtcttcac tgtctaggct tatttgtacc 1500
catccctctt agaaaggctt tccagatatt tgaaagtact tggatattgt gatgtaagct 1560
gtatctgatt tagagagcac cccaatccca gtaacactgt tttcttgcag actcatggag 1620
gtactacctt gatggtcttg gaaaagatcc agaagaattc tctgcattat caggcagaga 1680
gactcctgtt ccattctgtt actttcttcc aagcaaaaag agtctcactc tccttgttct 1740
gagccacata aagccggcgg tggagtgaca caagcatccc tgtggccacc accactatga 1800
ctgcattggg tcagacctga agccagcaca gcagtggggc ttgccaaggc tggctgtaac 1860
cacttcctgg ctactgccta tgtttgctga aggccctggg gctctacaat tagctggtgg 1920
caaagccggc caggcctgtg tccttccctt cagagccgtg aggtccccca ggccccaggt 1980
gagtccagag gtgccttttg ggaatcaggg actagagtaa aaaaacttgg aagtctagct 2040
ggtgttctat tgtactgtgg ctgagctggt attcagacca ccagatgcag tccttcccac 2100
tcttcctttc ccttcccaaa tgcagaggag cctcacccca tagccactgt caacccaggc 2160
cacgaggagt actgccaggc tacctccaat attcccttaa gcatcgaggt atcttaagtc 2220
aagcttgtgt tgaatgcttc ctgccttggg actcgccctt cagggcaatg ggttcccctc 2280
tggcctgggg caggtgcaga aatggtgtcc aacagtcaag tcctggaatc ggagacctca 2340
agagcccact tggtgcttta ccccactgtg gctgtgctgg tacctaaggt gcaagacaaa 2400
gccctcttta cttttccttc tgcttttctc aaacagaagg agttttgcct tgttgccacc 2460
acagctagta atgtgctgag tctcacctga agccagcaag tctcagaggc tcaaccaagg 2520
gccttgacat agtacctaaa tgtaactgct ggttattcag ggtcctgcca accttcagtt 2580
agcaggtgat gaatgctgcc aggactgagt ccttttcttc aaggtagtgg gtttccttct 2640
gacccatggt gtgtctagaa atgtcatctg ggaactaggg cctggaatgg gggcctcacg 2700
actcttccca gtcccctatt ctgccgcagc tgaactggta tccaagatgc aagatgaagt 2760
cctccccact cttctttctc ctctccacaa gcagaaggaa agggtctcct ttggagccac 2820
gggctgtgca gcctggagtt agggaagggg tgacgccagc acgcccttgg ctgccttagc 2880
tggtgtctca gtatgtcacc tgccctccca gttaactgcc tctgggccta gttcagcact 2940
agaactcgcc tgagatctgc agtcttcatg gcctgggctg cctttcaagt ttacttggag 3000
acccagactg tagccctcgg tggtgaggtt tgcaggaact caagtttaga ccactgggat 3060
cagtgattcc cctctgggta gggctgttat taaatgctcc ctccatgggc aggcgtcagc 3120
tgagttgggt gtggttttcc ttcctgccgt aacaggacag cactgagttc agtgcatcac 3180
agttgctgtg ttctccctcc cgtgcacgca gagatatcct cagtaccaca acacagctgc 3240
aggggcagag gggcgtcagt gattcaggac tgtttttgct atttcttcgg tgtctctttc 3300
agcattataa agttaagagc aggtactatg agtgctcacc tgatttttaa cacctcatga 3360
agttgttaaa ttggtgtcct tgtggagggg gatgatagct gcagtcttct attcctccat 3420
ttttctctgt ctcctcattt tttttttttt ttttttggag acggagtctt gctctgtcac 3480
ccaggctgga gtgcaatggc acgatctcgg ctcactgcag cctccgcctc caggttcaag 3540
cgattctcct gcctcagact cctgagtagc tgggactaca ggtgctacca tgaacagcta 3600
aatttaattt ttttaattat tatatattgt gggcgaagga ttacctaggt gccgaggcaa 3660
gagactaaag gcacaaactg tttcagtata ataaagaaaa tagaataaga atagtcataa 3720
tacaaattag atatagagat gatcgtggac aattatcaat cattattata aacattatta 3780
atcattagct tttaatatta ctctttgttg cattactaat ataacctagg aataaccagc 3840
gggtataggg tcaggtgctg aaagcacatt gtgacaagtt acctagaagg caagagctga 3900
gccttctgtc acgcccgcat aagggccgct tgagggctcc ttggtcaagc ggtaatgcca 3960
gtgtctggga agacacccac gacgtagcag accgcaaaag ggagtctcct ttccttggtg 4020
gagtcaggga acactctgct ccaccagctt cttgtgggag gctggatgtt atccaggcct 4080
gcccacagtt atccggaggc ctaaacccct ccctgtggtg ctgtgcttca gtggtcacgc 4140
tccttgtcca ctttcatgct cctccagcac tcctggttcc tctttgaagt tcgtagtaga 4200
taacggtaga agaaatagtg aaaggcttaa agtctttgac ctttcttata agtgcataga 4260
agaaaacgct gatgtatgcc accttctctc tgcttcagct acctaaaagg gaagggcccc 4320
ctgtcctatg atcacatgac ttgcttcaac ttgtcaatca cttagaagat tcaccctcct 4380
taccctgccc cctggtcttg tatgcaataa atatcagcgt gcccagccat tcgggaccac 4440
tacctgtctc cgtgtcttaa tggtagtggt cccctgggcc cagctgtttt ctctttatct 4500
ctttgtcctg tgtctttatt tattacaatc tctcatctcc gtacatggcg agaacacctg 4560
ctaagccccg taggcctcaa ctctacaata tatttatttt atattgccca ggttagtccc 4620
taactcctgg gttcaagtga tcctcccacc tcgacctccc aaagtgctat aattacaggt 4680
atgagccaca ccacacccgg ccaataatac acgtatcttt gcaagcataa acattcaggt 4740
atcctaacaa cagtctcata agtataagag ttatgagtag atgaactgta ttcataaaga 4800
aataggccaa aaagtaaaat gtattaacat atatgattgg taattgtgtg cactcaactt 4860
tataactgtg gtcatctgaa ataccaagat ggacaatata agtcttttga caaaatcaat 4920
aaaaatccgc agtgagttac caccacacat gcagttgccc aaaaagccaa gatctcaaaa 4980
tattttatct ttcacaaata tggatgtaca aaaagaagat attttattga ggaagttgca 5040
atgtttttac atacatgcac aatgcttaca caaattcagc ttcgtgataa tgcactttca 5100
tggagtcaaa tttgcaaaaa tgcataaaat gaattagaac tttctaaaca tctttataca 5160
atttatacct tcagtattaa aaatggactg aggaggccgg gcacagtggc tcacacctgt 5220
aatccctgac tttgagaggc tgaagtgggc ggatcacttg aggttaggag ttggagacct 5280
gcctggccaa catagtgaaa ccctgtctgt agtaaaaata caaaaattaa cacttgagcc 5340
caggaggcgg aggttggagt gagctgagat tgtaccactg cactccagcc tgggtgacaa 5400
agtgagaccc tgtctcaata aaaaataaaa ataaaaaaag tatgttatta tttcctgttt 5460
tattgtctaa aatggtctat gaagtgctct gttgtgtttt tatgtttctc aaatctcctt 5520
taaaaaatat aaataaagaa ttttaaagaa tttttaaaat tattttgtca agaattatat 5580
tttcaggatt ttaatcattt gggattggaa ttttggggat tttggacttc agggattttg 5640
atctttcagg atttcatcat ttgggattat aacattcaag attgtgtctt ttgggattat 5700
ggcccaaacc catgcaatgg gccagttctt tgaaaggaat agtgttccat aactcacaca 5760
aggagaaata gaaaatttga ataggcctat acctgttaaa ctaaatcaat gatttgccaa 5820
agaggtaata aggtttggac tacatttatg tcagcaaaga ggagaggaca gattaattca 5880
ttcaataagt gattgagtag atacctactg gtgtcaggca ttgttctagg cactgggaat 5940
acagcagcaa acaaaatgat tgagtagata cctactggtg tcaggcattg ttctaggcac 6000
tgggaataca gcagcaaaca aaatagatca ccccttgggg ctttcaagtg gagtatcggt 6060
gacagagtgg gtgtctgatt ggatatcggg gaaaaggaga aaggtgcgat gaggcaattc 6120
aggggtcatt ggtacctgga tttttcacct tgaacaacta agtgatatca ttggacttgt 6180
tgggtagaca aggtctaggg gacatccaga tggactcatt ttcacagcta gcaagagaga 6240
cattctgaag atcaatagta agggagaaac tggaagtaca gactttgggg tcctaagagg 6300
cagttagaac catgggagta gatgattgta taagataaat agttttgcct cttacttccc 6360
ccaccccaac aaatgaaatg gaatcagaag tgtaggagga gaactagggg aaattaggaa 6420
gtgagatttt catcagggca aaatgatgct gacggatcaa atgggacaag aaatgtgaaa 6480
aaaatcattt tacactttta tccatctatt gatggataaa atttaaccaa aattgagtca 6540
ctgaatttga tcattgatca aatttttctt actctgataa gagttttcag aaaactctga 6600
tcagagtttt cttactctga tatctcatat tcaaaaggga aagaaaatca acaacacaat 6660
aaacaacctc ctgaggaatg cagtgtaaaa gcaaggcttg gtgtgttgtt tcgcaaaaaa 6720
tcttgctttg tcagcattaa tactaacaga taacatgaga agaggcaaaa gtgttggcta 6780
aatgtaccat aactgcctgg gaatagttat ctgtagtcta tttttttttt ttctcatcta 6840
aagttcttag atgaaagaga gctgggttac tgtgaaccaa aatctcctgc t 6891
<210> 2
<211> 21
<212> DNA
<213>The primer sequence F of specific amplification lncRNAsT267384
<400> 2
tctgggcatt gaagagtgaa g 21
<210> 3
<211> 22
<212> DNA
<213>The primer sequence R of specific amplification lncRNAsT267384
<400> 3
agagggatgg gtacaaataa gc 22
Claims (9)
1. molecular marked compound lncRNAsT267384, sequence such as SEQ ID NO for diagnosis of glaucoma:Shown in 1.
2. application of the product of lncRNAsT267384 expressions in preparing diagnosis of glaucoma tool is detected,
The sequence of lncRNAsT267384 such as SEQ ID NO:Shown in 1.
3. application according to claim 2, which is characterized in that the system of the detection lncRNAsT267384 expressions
Agent includes：The preparation of lncRNAsT267384 expressions is detected by real-time fluorescence quantitative PCR.
4. application according to claim 3, which is characterized in that detect lncRNAsT267384 with real-time fluorescence quantitative PCR
The preparation of expression includes the primer of specific amplification lncRNAsT267384.
5. application according to claim 4, which is characterized in that detect lncRNAsT267384 with real-time fluorescence quantitative PCR
The primer sequence of the specific amplification lncRNAsT267384 of expression is：
F:5'- TCTGGGCATTGAAGAGTGAAG-3 ',
R:5'-AGAGGGATGGGTACAAATAAGC-3’。
6. a kind of kit for diagnosis of glaucoma, which is characterized in that include detection lncRNAsT267384 expressions
Reagent, the sequence such as SEQ ID NO of lncRNAsT267384:Shown in 1.
7. the kit according to claim 6 for diagnosis of glaucoma, which is characterized in that comprising fixed by real-time fluorescence
Measure the reagent of PCR detection lncRNAsT267384 expressions.
8. the kit according to claim 7 for diagnosis of glaucoma, which is characterized in that described passes through real-time fluorescence
The reagent of quantitative PCR detection lncRNAsT267384 expressions includes to pass through real-time fluorescence quantitative PCR specific amplification
The primer of lncRNAsT267384.
9. the kit according to claim 7 or 8 for diagnosis of glaucoma, which is characterized in that described by real-time
The reagent of fluorescence quantitative PCR detection lncRNAsT267384 expressions includes a pair of by real-time fluorescence quantitative PCR specificity
The primer of lncRNAsT267384 is expanded, primer sequence is：
F:5'- TCTGGGCATTGAAGAGTGAAG-3 ',
R:5'-AGAGGGATGGGTACAAATAAGC-3’。
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| CN201710423197.3A CN107142316B (en) | 2017-06-07 | 2017-06-07 | Diagnosis of glaucoma molecular marked compound lncRNAsT267384, kit and application |
| US16/619,945 US20200181705A1 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers, kits and applications for glaucoma diagnosis |
| PCT/CN2018/088324 WO2018223848A1 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers for diagnosis of glaucoma, kit, and application |
| EP18813073.6A EP3643794A4 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers for diagnosis of glaucoma, kit, and application |
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| CN201710423197.3A CN107142316B (en) | 2017-06-07 | 2017-06-07 | Diagnosis of glaucoma molecular marked compound lncRNAsT267384, kit and application |
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| CN107142316A CN107142316A (en) | 2017-09-08 |
| CN107142316B true CN107142316B (en) | 2018-10-26 |
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| US20200190558A1 (en) * | 2017-06-07 | 2020-06-18 | The Second Xiangya Hospital Of Central South University | Three molecular markers, kits and applications for glaucoma diagnosis |
| US20210285047A1 (en) * | 2017-06-07 | 2021-09-16 | The Second Xiangya Hospital Of Central South University | Two molecular markers, kits and applications for glaucoma diagnosis |
| EP3643794A4 (en) * | 2017-06-07 | 2021-02-17 | The Second Xiangya Hospital of Central South University | Two molecular markers for diagnosis of glaucoma, kit, and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770562A (en) * | 2010-05-26 | 2012-11-07 | 目立康株式会社 | Normal-tension glaucoma susceptibility gene and method for using the same |
| CN105002182A (en) * | 2014-11-18 | 2015-10-28 | 南京医科大学眼科医院 | Application of LncRNA-GAS5 in preparing glaucoma diagnosis reagent |
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2017
- 2017-06-07 CN CN201710423197.3A patent/CN107142316B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770562A (en) * | 2010-05-26 | 2012-11-07 | 目立康株式会社 | Normal-tension glaucoma susceptibility gene and method for using the same |
| CN105002182A (en) * | 2014-11-18 | 2015-10-28 | 南京医科大学眼科医院 | Application of LncRNA-GAS5 in preparing glaucoma diagnosis reagent |
Non-Patent Citations (1)
| Title |
|---|
| Novel Insights into the Role of Long Noncoding RNA in Ocular Diseases;Fang Li et al.;《Internationl Journal of Molecular science》;20160331;第17卷(第478期);第1-11页 * |
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