CN106978509A - Diagnosis of glaucoma molecular marked compound lncRNAs ENST00000607393, kit and application - Google Patents
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Abstract
The invention discloses molecular marked compound lncRNAs ENST00000607393, kit and the application for diagnosis of glaucoma.It is found by the applicant that relative to control group crowd, lncRNAs:ENST00000607393 equal up-regulated expressions in the aqueous humor, iris and serum of glaucoma patient.Prompting ENST00000607393 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, with far-reaching clinical meaning and generalization.
Description
Technical field:
The present invention relates to molecular diagnostic techniques field, and in particular to a kind of to contribute to the long-chain non-coding of diagnosis of glaucoma
RNA marks, kit and application.
Background technology:
Eyes are the highly important sense organs of human body, can receive the light stimulus of outside, and light impulsion is sent to greatly
Mesencephalic centre and cause vision.Leonardo da Vinci once said：" the eyes are the windows of the mind, by eyes, and 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, people by sense organ from the information that the external world is obtained, greatly
About 90% is completed by eyes.The data of the World Health Organization shows that ophthalmology disease turns into after tumour, angiocardiopathy
The 3rd harm afterwards and the disease of influence people's life quality.In all ophthalmology diseases, glaucoma is then that first place can not
The blinding illness in eye reversed, the reason for it influences visual quality is to cause to lose by threatening and damaging optic nerve and its path
It is bright, so as to seriously threaten the Vision Health of the mankind, loss difficult to the appraisal is caused to personal, family and society.
The main harm that glaucoma is caused is influence visual performance, even in developed country, also only 50% or so
Glaucoma patient timely can be diagnosed and be treated, moreover, the pathogenic factors of glaucoma, genetics law etc. fail to understand
, therefore we will scientifically grasp its regularity of occurrence and development, so as to be early diagnosed and early treatment, it is to avoid glaucoma is suffered from
The blindness of person.At present, the inspection in terms of the diagnosis of glaucoma mainly relies on medical history, morphology and function assessment, such as tonometry,
Ultrasound biomicroscopy UBM, eye-ground photography, optical coherence tomography and perimetry etc..Although these inspections can diagnose green light
Eye, but research shows that the glaucoma diagnosed by morphology and function assessment means, the infringement of patient visual's function is alreadyd exceed
50%.And glaucoma related biochemical analysis, serological screening and examination criteria be still in relative space state, therefore find one
The mark of high sensitivity and high specific is planted to diagnosis of glaucoma and is monitored particularly important.
Genome plan research shows, in 3,000,000,000 base-pairs of composition human genome, only 1.5% nucleotides
Sequence is used for protein coding, and remaining 98.5% genome is non-protein coded sequence.These sequences were once considered as to enter
" determined garbage sequence " accumulated during change 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
Into RNA, wherein nearly 74% transcription product is non-coding RNA (non-coding RNAs, ncRNAs).In ncRNA, sequence
The transcript that length is more than 200 bases is long-chain non-coding RNAs (long noncoding RNA, lncRNAs), they by
Received much concern in rich and mechanism of action the diversity of function.LncRNAs can transcription, transcription after and translate it is multiple
The expression of protein coding gene is adjusted in aspect, so as to widely participate in the important life including cell differentiation and body development
Life process, generation of its unconventionality expression also with a variety of major diseases of the mankind is closely related.
LncRNA has well-conserved and tissue specificity, is enriched very much in brain content.LncRNA not only take part in
Growing and perfect in shape and function for nervous system, makes nervous system be carried out according to regular hour order and in certain space
Growth and development, and participate in performing the function of nervous system.LncRNA participates in the development of nervous system by number of mechanisms
And during function is performed, including it is used 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 degradeds and translational control.Therefore, the composition and expression for detecting lncRNAs are utilized
Change come the physiology and pathological state that reflect nervous system be feasible means.
It is tolerant that aqueous humor belongs to intraocular, is produced by ciliary body, and blood is eventually entered into by pupil, trabecular network etc., and in dynamic
Among state circulation, its composition constitutes closely related with eyeball local physiological and pathology environment.Excretion body is most important as body
Intermediary's material for mutually exchanging of iuntercellular, contain lncRNAs, mRNAs, protein and lipid etc..Included in excretion body
Material 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 correlation
LncRNAs researchs are with the potential provided fundamental basis for the detection of glaucoma associated biomarkers.
The content of the invention
An object of the present invention is to provide a kind of long-chain non-coding RNA early diagnosed for glaucoma, to glaucoma
Morbidity detection it is significant.
The molecular marked compound lncRNAs ENST00000607393, its sequence such as SEQ ID early diagnosed for glaucoma
NO:Shown in 1.
The second object of the present invention is to provide above-mentioned molecular marked compound lncRNAs ENST00000607393 application.Inspection
Survey application of the product of lncRNAs ENST00000607393 expressions in glaucoma early diagnosis instrument is prepared.
The product of described detection lncRNAs ENST00000607393 expressions includes：Pass through real time fluorescent quantitative
PCR detects the preparation of lncRNAs ENST00000607393 expressions.
The preparation for detecting lncRNAs ENST00000607393 expressions with real-time fluorescence quantitative PCR includes specificity
Expand lncRNAs ENST00000607393 primer.
The specific amplification of lncRNAs ENST00000607393 expressions is detected with real-time fluorescence quantitative PCR
LncRNAs ENST00000607393 primer sequence is：
F:5'-GGCGTCTGTTTGTTTATTGGC-3 ',
R:5'-TCACTTTTGCTTGGGGAGATT-3’。
The third object of the present invention is to provide a kind of kit early diagnosed for glaucoma, includes detection lncRNAs
The reagent of ENST00000607393 expressions.Specifically include and lncRNAs is detected by real-time fluorescence quantitative PCR
The reagent of ENST00000607393 expressions.
The described reagent bag that lncRNAs ENST00000607393 expressions are detected by real-time fluorescence quantitative PCR
Containing the primer by real-time fluorescence quantitative PCR specific amplification lncRNAs ENST00000607393.
The described reagent bag that lncRNAs ENST00000607393 expressions are detected by real-time fluorescence quantitative PCR
Primer containing a pair by real-time fluorescence quantitative PCR specific amplification lncRNAs ENST00000607393, its primer sequence
For：
F:5'-GGCGTCTGTTTGTTTATTGGC-3 ',
R:5'-TCACTTTTGCTTGGGGAGATT-3’。
We exist with the checking 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 three kinds are organized,
Aqueous humor has highest diagnostic significance, and sensitiveness/specificity that three kinds of lncRNAs diagnose glaucoma in aqueous humor is all higher than
90%.The possible cause of this phenomenon is due to the presence of blood aqueous barrier, significantly limit haematogenous immunocyte and comes from
The signaling molecule at other positions of whole body enters aqueous humor.Therefore compared with serum for, aqueous humor specific can more represent the physiology of eye
And pathological state.Therefore, aqueous humor may have bigger diagnosis of glaucoma 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:ENST00000607393 is in green grass or young crops
Equal up-regulated expression in aqueous humor, iris and the serum of light eye patient.Prompting ENST00000607393 is the high expression in glaucoma
LncRNAs, contributes to the biomarker of diagnosis of glaucoma.The present invention provides strong molecule life for diagnosis of glaucoma
Thing basis, with far-reaching clinical meaning and generalization.
Brief 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
Analysis of spectrum 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 diagram expressed in water；D-F：T267384, ENST00000607393 and T342877 are white in glaucoma and age related
The case figure expressed in cataract or glaucoma 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 diagram 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 diagram；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 is diagnosed in aqueous humor using T267384, TG-AUC is 0.998；B：Utilize
ENST00000607393 diagnoses the ROC curve of glaucoma in aqueous humor, and TG-AUC is 0.998；C：Existed using T342877
The ROC curve of glaucoma is diagnosed in aqueous humor, TG-AUC is 0.983；D：Using ENST00000607393 in iris tissue
The ROC curve of glaucoma is diagnosed, TG-AUC is 0.793；E：The ROC for diagnosing glaucoma in serum using T267384 is bent
Line, TG-AUC is 0.620；F：Diagnosed using ENST00000607393 in serum under the ROC curve of glaucoma, curve
Area is 0.638.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1：LncRNAs and mRNAs express spectra in glaucoma patient aqueous humor：
1.1 materials and reagent
1.1.1 key instrument
Conventional centrifuge and refrigerated centrifuge are purchased from Eppendorf companies；Supercentrifuge is purchased from Beckman companies；
Real-Time PCR instruments device is 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 prosperous 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 kit are purchased from Agilent Technologies companies；People lncRNAs microarray
Chip is purchased from Arraystar companies.
1.1.3 reagent is configured
Various Western blotting agents useful for same according to《Molecular cloning (third edition)》The method configuration of middle offer.
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, undiluted room is extracted before formal operating procedure starts using 1ml disposable sterilized injector in corneal limbus
This 0.1ml of water gauge, injects in autoclaved 0.5mlEP pipes immediately, be placed in -80 DEG C of low temperature refrigerators be kept in dark place it is standby.It is all
Selected patient is to be in hospital to need the glaucoma patient and patients with senile cataract that carry out operative treatment in our hospital.Include mark
It is accurate：1. primary open-angle glaucoma：A. intraocular pressure>21mmHgB. glaucomatous disks are damaged and/RNFL defects C. is typically blue or green
Light eye property defect of visual field D. anterior chamber angles are opened.With four above or with A therein, D, B or C person.2. normal tension is blue or green
Light eye：With the optic disk change similar to POAG, RNFL and visual field damage, 24h tonometries≤21mmHg, room angle is opened.
3. primary angle-closure glaucoma：Optic disk with glaucoma changes, RNFL and visual field damage, intraocular pressure>21mmHg, anterior chamber angle becomes
Narrow or 4. age-related cataracts of closing (control group)：Crystalline lens in cortex and/or caryogram is muddy and/or rear capsule under property mix
It is turbid.Exclusion standard：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
False negative rate>25%.5. age-related cataract：Exclude concurrency, traumatic, congenital cataract and exclude with other
Ophthalmic disease person, exclusion overripe stage case.
1.2.2 RNA is extracted
Take aqueous humor 0.1ml to add 0.5mlTrizol reagents, acutely add 0.1 milliliter of chloroform after concussion.And after through centrifugation,
10 microlitres of DPEC water are dissolved in after precipitation and the cleaning of 75% ethanol, through each pipe RNA concentration of Q5000 apparatus measures.
1.2.3 RNA amplification
Use TargetAmpTM1-Round aRNA Amplification Kit 103 (epicentre) press raw manufacturer and said
Step shown in bright book is expanded to RNA.Substantially step is as follows：Synthesize single into single-stranded cDNA according to RNA samples first:RNA is miscellaneous
Product is handed over, the use of RNase H enzymes is then small fragment sequence by the digestion of above-mentioned hybrid product, these small fragment sequences can assist double
Chain cDNA synthesis.It is last that antisense RNA is synthesized by double-strand cDNA transcriptions.
1.2.4 cDNA is synthesized and marked
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 IMAQ and data analysis
The fluorescence intensity of chip is scanned using GenePix 4000B chip scanners, and experimental result is converted into numeral
Type data are preserved.P values<0.05 is that difference is statistically significant.
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, the lncRNAs of 2 times of up-regulated expressions is 4372 kinds, 2 in glaucoma patient aqueous humor
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
Because its easily taking property, individual specificity and relative other organ by body is influenceed less feature, aqueous humor is a kind of
The larger research object of value of eye disease biomarker correlative study.Although also, a variety of research reports existing at present
The related epidemiology of glaucoma and science of heredity hazards, but the correlative study using aqueous humor as research object is relatively fewer.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 problem, we increase RNA amplification step before chip hybridization is carried out, so with guarantee
Subsequent step is smoothed out.By using lncRNAs chip microarray analysis methods, 11728 kinds of lncRNAs and 6686 kinds
MRNAs is detected in 10 parts of Aqueous Humor of Glaucomas samples, in this age-related cataract patient aqueous humor lncRNAs and
There is mRNAs express spectras lncRNAs and mRNAs express spectras in significant difference, therefore aqueous humor to show individual specificity and disease
Specificity.As far as we know, this is first Aqueous Humor of Glaucomas correlation lncRNAs and mRNAs express spectras research.
Embodiment 2：The correlation of specific lncRNAs and mRNAs in clear and definite aqueous humor is analyzed by CNC
Further to inquire into the lncRNAs expressed in glaucoma patient aqueous humor possibility function, we use CNC
(coding-noncoding gene co-expression) analysis clearly has with the mRNAs expression of glaucoma related gene
The lncRNAs of significant correlation.CNS analyses are a kind of by lncRNA and mRNA coexpression data, and lncRNA and mRNA is joined
The analysis method that system gets up.Analyzed by CNC it can be found that there is the mRNA of identical expression pattern with some lncRNA, by this
A little mRNA function, can connect lncRNA with signal specific path or disease condition, consequently facilitating prediction lncRNA
Function, and disclose its mechanism of action.
2.1 method
Select differential expression in aqueous humor and occur mRNAs of the development with correlation with glaucoma, these mRNAs are existed
Expression data in different glaucoma patient's aqueous humor are averaged；Seek the data and green light after the mRNAs standardization picked out
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 instruments.
2.2 result
For age-related cataract, in glaucoma patient aqueous humor differential expression and with glaucoma occur development tool
The mRNAs for having correlation is following ten kinds：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), jaw albumen E3 (forkhead box E3, FOXE3), secretagogue receptor
(growth hormone secretagogue receptor, GHSR), jaw PROTEIN C 1 (forkhead box C1,
FOXC1), cross-film and coiled-coil domain 1 (transmembrane and coiled-coil domains 1, TMCO1),
PH 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 that these mRNAs are expressed has
10 kinds.
Embodiment 3
Our 10 kinds using qRT-PCR in glaucoma and age related cataract patient aqueous humor in checking embodiment 2
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 differential expression amount most significant 3 kinds of T267384,
ENST00000607393 and T342877, is further verified.
Embodiment 4
We with qRT-PCR detection methods checking 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
Conventional centrifuge and refrigerated centrifuge are purchased from Eppendorf companies；Supercentrifuge is purchased from Beckman companies；
Real-Time PCR instruments device is 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 prosperous 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 is configured
Various Western blotting agents useful for same according to《Molecular cloning (third edition)》The method configuration of middle offer.
4.2 method
4.2.1 aqueous humor, serum and iris sample prepare
Aqueous humor samples step as described in 1.2.1 prepares.Serum sample is derived from glaucoma patient group and healthy control group crowd,
Wherein glaucoma patient include and exclusion standard is with described in 1.2.1, control group serum be derived from glaucoma groups patient age with
The healthy population of sex matching.Iris sample is derived from glaucoma patient group and control group, and wherein glaucoma patient being included and arranging
Except standard is with described in 1.2.1, control group iris sample is derived from refined two hospital in the Hunan matched with the glaucoma patient age with sex angle
Film donates the iris tissue of volunteer.
4.2.2 RNA is extracted
0.1 milliliter of aqueous humor or iris tissue add 0.5 milliliter of Trizol reagent, acutely add 0.1 milliliter of chloroform after concussion.
And after through centrifugation, precipitation and 75% ethanol cleaning after be dissolved in 20 microlitres of DPEC water, through each hole RNA concentration of Q5000 apparatus measures.Outside
All venous blood is put into centrifuge after standing 30 minutes, 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 by RNA reverse transcriptions into cDNA according to raw manufacturers instruction first, then expanded with PCR method
CDNA, the product amount of quantitative amplification is detected by determining 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, compare progress Mann-whitney U inspections.P values<0.05 is difference
It is statistically significant.
4.3 result
Exist further to expand aqueous humor samples amount checking correlation lncRNAs expression 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, the patients with senile cataract aqueous humor sample that 30 ages match with sex), 50 iris tissues (40
Glaucoma patient iris tissue sample, the cornea that 10 ages match with sex donates the iris tissue of volunteer) and 158 blood
In final proof sheet (103 glaucoma patient serum samples, the healthy population serum sample that 55 ages match with sex)
T267384, ENST00000607393 and T342877 expression.As a result show, in two class aqueous humor samples, three kinds
LncRNAs expression quantity has significant difference, and its expression quantity in glaucoma patient aqueous humor is followed successively by white in age related
In cataract or glaucoma patient's aqueous humor expression quantity 4.4036,2.1467 and 2.9692 times；The ENST00000607393 in two class iris tissues
Expression quantity there is significant difference, its in glaucoma patient iris tissue expression quantity be in control group iris tissue express
3.3436 times of amount, and T267384 and T342877 expression quantity no difference of science of statistics；In two class serum samples, T267384
And ENST00000607393 expression quantity has significant difference, its expression quantity in glaucoma patient serum is followed successively by right
According to 1.2878 and 1.5301 times of expression quantity in group serum, and T342877 expression quantity no difference of science of statistics (table 1,2,3) (figure
4,5,6).To further appreciate that these three lncRNAs diagnose the validity of glaucoma 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
0.998th, 0.998 and 0.983.ENST00000607393 diagnoses area under the ROC curve of glaucoma in iris tissue
0.793；T267384 and ENST00000607393 diagnose Line Integral under the ROC curve of glaucoma in serum sample
0.620 and 0.638.Be set to 1.5437 when the diagnostic value of T267384, ENST00000607393 and T342877 in aqueous humor,
When 1.1485 and 2.1052, its sensitiveness/specificity diagnosed 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 to 0.2470, its sensitiveness/specificity diagnosed
For 0.700/0.700；When the diagnostic value of T267384 and ENST00000607393 in serum is set to 0.7191 and 0.8376,
Its sensitiveness/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 Sensitivity 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 lncRNAs ENST00000607393, kit and application
<130>Nothing
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 312
<212> DNA
<213> lncRNA：ENST00000607393 sequence
<400> 1
ctagcttccg agggacttct ctgcgagcac cgcaggcgtg tgcatttctt cgaatctgag 60
aattccagcg aactcgggct ctaccagagt ttaagtcggc ccctggggcg tctgtttgtt 120
tattggctct taagggaaaa aagtttgaag atctttaacc agcctcagac ctggcgcggg 180
cggaaatccc ggaatctccc caagcaaaag tgatataact tcaagtcgtt atataagtgg 240
ggggctcctt cttcccctcc ctacgccgag ccttctcatt atcattccgg gagtaaaatg 300
tgtctcgctc aa 312
<210> 2
<211> 21
<212> DNA
<213>Specific amplification lncRNAs ENST00000607393 primer sequence F
<400> 2
ggcgtctgtt tgtttattgg c 21
<210> 3
<211> 21
<212> DNA
<213>Specific amplification lncRNAs ENST00000607393 primer sequence R
<400> 3
tcacttttgc ttggggagat t 21
Claims (9)
1. molecular marked compound lncRNAs ENST00000607393, its sequence such as SEQ ID NO for diagnosis of glaucoma:1 institute
Show.
2. detect application of the product of lncRNAs ENST00000607393 expressions in diagnosis of glaucoma instrument is prepared.
3. application according to claim 2, it is characterised in that described detection lncRNAs ENST00000607393 tables
Include up to horizontal product：The system of lncRNAs ENST00000607393 expressions is detected by real-time fluorescence quantitative PCR
Agent.
4. application according to claim 3, it is characterised in that detect lncRNAs with real-time fluorescence quantitative PCR
The preparation of ENST00000607393 expressions includes specific amplification lncRNAs ENST00000607393 primer.
5. application according to claim 4, it is characterised in that detect lncRNAs with real-time fluorescence quantitative PCR
The specific amplification lncRNAs ENST00000607393 of ENST00000607393 expressions primer sequence is：
F:5'-GGCGTCTGTTTGTTTATTGGC-3 ',
R:5'-TCACTTTTGCTTGGGGAGATT-3’。
6. a kind of kit for diagnosis of glaucoma, it is characterised in that include detection lncRNAs ENST00000607393 tables
Up to horizontal reagent.
7. the kit according to claim 6 for diagnosis of glaucoma, it is characterised in that comprising fixed by real-time fluorescence
Measure the reagent that PCR detects lncRNAs ENST00000607393 expressions.
8. the kit according to claim 7 for diagnosis of glaucoma, it is characterised in that described to pass through real-time fluorescence
The reagent of quantitative PCR detection lncRNAs ENST00000607393 expressions is included by real-time fluorescence quantitative PCR specificity
Expand lncRNAs ENST00000607393 primer.
9. the kit for diagnosis of glaucoma according to claim 7 or 8, it is characterised in that described by real-time
The reagent of fluorescence quantitative PCR detection lncRNAs ENST00000607393 expressions passes through real time fluorescent quantitative comprising a pair
PCR specific amplification lncRNAs ENST00000607393 primer, its primer sequence is：
F:5'-GGCGTCTGTTTGTTTATTGGC-3 ',
R:5'-TCACTTTTGCTTGGGGAGATT-3’。
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| CN201710423729.3A CN106978509B (en) | 2017-06-07 | 2017-06-07 | Diagnosis of glaucoma molecular marked compound lncRNAs ENST00000607393, kit and application |
| PCT/CN2018/088322 WO2018223846A1 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers for diagnosis of glaucoma, kit, and application |
| EP18812953.0A EP3636772A4 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers for diagnosis of glaucoma, kit, and application |
| US16/619,946 US20210285047A1 (en) | 2017-06-07 | 2018-05-25 | Two molecular markers, kits and applications for glaucoma diagnosis |
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| CN201710423729.3A CN106978509B (en) | 2017-06-07 | 2017-06-07 | Diagnosis of glaucoma molecular marked compound lncRNAs ENST00000607393, kit and application |
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| CN106978509A true CN106978509A (en) | 2017-07-25 |
| CN106978509B CN106978509B (en) | 2018-10-26 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018223846A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Two molecular markers for diagnosis of glaucoma, kit, and application |
| WO2018223849A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Three molecular markers for diagnosis of glaucoma, kit, and application |
| WO2018223847A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Application of lncrnasenst00000607393 sirna in preparing preparation for treating glaucoma |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018223846A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Two molecular markers for diagnosis of glaucoma, kit, and application |
| WO2018223849A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Three molecular markers for diagnosis of glaucoma, kit, and application |
| WO2018223847A1 (en) * | 2017-06-07 | 2018-12-13 | 中南大学湘雅二医院 | Application of lncrnasenst00000607393 sirna in preparing preparation for treating glaucoma |
| US11160824B2 (en) | 2017-06-07 | 2021-11-02 | The Second Xiangya Hospital Of Central South University | Application of LNCRNAS ENST00000607393 siRNA in preparing preparation for treating glaucoma |
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| CN106978509B (en) | 2018-10-26 |
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