CN109988830A - A kind of lncRNA and its application for sepsis markers - Google Patents
A kind of lncRNA and its application for sepsis markers Download PDFInfo
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Abstract
The present invention provides a kind of lncRNA for sepsis markers and its application, the lncRNA is lncRNA ENST00000490593.1, and nucleotide sequence is as shown in SEQ ID NO.1.Expression of the present invention by detection lncRNA ENST00000490593.1 in the sample, further enriches the research of pathogenesis of sepsis mechanism, is of great significance to pyemia early diagnosis.
Description
Technical field
The invention belongs to field of biomedicine, it is related to a kind of non-coding RNA and its application, more particularly to a kind of is used for septicopyemia
The lncRNA of disease marker and its application.
Background technique
Pyemia is a kind of typical complex disease, is to show patient to the bad clinical state of infection reaction.Pyemia
Status with high incidence, high mortality and high medical expense.Early diagnosis is to reduce the best plan rate of pyemia case fatality rate.
Clinical existing sepsis diagnosis standard still relies primarily on clinical symptoms, and the clinical symptoms of Systemic inflammation may
It partly overlaps with non-infection critical condition (such as wound, burn, pancreatitis, autoimmunity disease and graft-rejection), microorganism
Culture is also shown as negative often because of early period or adjoint antibiotherapy, causes largely to diagnose delay.With transcript profile
It learns and the continuous development of genomics technologies, non-coding RNA (non-coding RNAs, ncRNAs) becomes research hotspot.It is numerous
Research confirms that ncRNAs plays important regulation role in cell differentiation and growth.Long-chain non-coding RNAs (long non-
Coding RNAs, lncRNAs) it be a kind of transcript length is more than 200nt, the not open reading frame of coding protein.In recent years
Come the study found that lncRNA can on epigenetic, transcription and post-transcriptional level controlling gene expression, with human diseases
There is substantial connection in occurrence and development and treatment, the research of most lncRNA is concentrated mainly on the neck such as tumour or neurogenic disease
Domain rarely has the report of lncRNA related with pyemia.
CN109022570A, which discloses LOC105372511, can be used as diagnosis of sepsis marker.Research shows that
The raising more significant than normal person of LOC105372511 content in the blood of sepsis patient.Therefore LOC105372511 can be used as
The molecular marker of diagnosis of sepsis disease.
CN109112212A discloses sepsis diagnosis relevant molecule.The present invention by sequencing screening in sepsis patient and
The molecule of expression is had differences in normal healthy controls crowd, research shows that content ratio of the LINC01871 in sepsis patient blood
Healthy People is low.It accordingly can be using LINC01871 as the biomarker of diagnosis of sepsis disease.
CN109022571A discloses LOC105369645 and is preparing the application in diagnostic products.The diagnostic products can be used for
Diagnosis of sepsis.By detect LOC105369645 level may determine that subject in the future suffer from pyemic risk or
Make a definite diagnosis pyemic state.
CN109055535A discloses purposes of the LOC101927627 as the molecular marker of diagnosis of sepsis disease.Study table
There are significant differences for expression of the bright LOC101927627 in sepsis patient blood and in normal healthy controls crowd, think accordingly
LOC101927627 can be used as the molecular marker of diagnosis of sepsis disease.
Therefore it provides a kind of lncRNA adjuvant clinical diagnosis of sepsis disease for sepsis markers, research pathogenesis
It is of great significance.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provide a kind of lncRNA for sepsis markers
And its application further enriches pyemia hair by the expression of detection lncRNA ENST00000490593.1 in the sample
The research of the interpretation of the cause, onset and process of an illness, is of great significance to pyemia early diagnosis.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of lncRNA for sepsis markers, the lncRNA is lncRNA
ENST00000490593.1, nucleotide sequence is as shown in SEQ ID NO.1.
The particular sequence of lncRNA ENST00000490593.1 is as shown in SEQ ID NO.1: CAGCACTTGGGATGTG
CACCAACAGTTCTTATGGGGGCCCGGCCTCCTCATCACTCCAGTTCTGGATGAAGTAAGTGTTCCCACAGAGATAC
ACTAGAGATCTCTGCATCTGTATCTGCTGCCCTGCAAACTCCTCTCTGCTTCTTATTCCAAACTCACTTCTGACAT
CTGTGACTGAGTCAGCCTTGAAGAGAGTGTGCTAGGTTTTGAGAAGCTAGACATCTGGTAACTACACATTTTTCTT
TCTAGAATTTCTTTTTTCTCTTGATCAAGTTCCCAACCAGTTTAGAAGCATCTACTGGAAACTCTAGAGTTACAAG
TTATGTTAGGTTGTTCTTGCATTGCTATACAGAAATTCCTGACACTGAGTGATTTACAAAGAAAAGAGATTTCATT
GGCTCACAGTTCTGAAAGCTTTACAAGAAGCATGATGCTGGTATCTGTGCAGCTTCTAGGGAGGCCTCAGGAAGCT
TACAGTCATGGAGGAAGGTGAAGAGGAGCAGCGATGTCATATAGCAAAAGCAGGAACAAGACGGGGTGGGGGGAGT
TGCCACACACTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCACA
ATCCCAGCTCACTGCAACCTCCAACTCCTGGGTTCACATGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGGTTA
CAGGCACCTGCCACCAAGCTCGGCTAATTTTGTATTTTTTGTAGAGACGGGGTTTCATGTTAGCCAGGCTGGTCTC
AAACTGCTGACCTCAGATGATCCACCTGCCTCAGCCTCCAAAGTGCTGAGATTACAGGTGTGAGTCACCGCGCTTG
GCCGCCACACACTTTTAAACAACCAAATCTTACAAGAATTCACTCACTATCACCAGGACAGAACCAAGGGAATGGT
GTAAACCATTCATGAGAAATCTGCCCTCATCATCCAGTCAGCTCCCTCCAGGCCCCACCTCCAACACTGGGGATTA
CATTTCAACATGAGATTTGGGCAGACAAACATCCAAACTATATCAAGAGTGGTCCTATTTGTTGTTGGATTCTCAT
AGCAGATCCTGACTTGCTCCATGACGTAGAAACAGGATGGAGCAGTGACCCAACACTCACTCTAAAGTCAGGCTGA
TCGGGATGTGATTGTTGTCTCTGCCACTTTGAGCCTGTGTGATCAGGGGTGACTATTGTGCTGGCTTCTCATCTGC
AGAGGGGTAATACTACTACTACTAAAAATAATGGTACCCACCATTTGGGAATGTTGGGAAAATTAAATGAGGTAAT
CATGTAAAGCTGGAATGTTTGTCACCACACATGTTTTATTATTATTTTATGATTATTATTATTCCTAATGTGGCTA
CGTTTGGGATTTCTCTCTGGGTTGCCAAGTTACAGTTTCTATTATTTGTATTAGAGGAGAAGCAGAAGAAAGGCAT
AATAGCAGGGCATTCCTGCCCTCTCTGTGTATGATTAAGAAATTCCCTAGAGAATAAAAAGAAATCCATTTTATTT
CCCTCAGGGGACACTACATTTTTTTAATTTCAGGGTGCAGAGAAAGTGATGGCATATGTGCCTGATGCTGTCTGGT
ATGACTACGAGACTGTAAGTAGCTTTGACTTTTCTTCTACTCCTTAAGACTGTAGCTGCAGCTGCATAGACAAGCT
ACCTTTCTGGAGAGAGAAACATCCATCTACATGCTGAGGAGTAGTTTTTAGTGTTTTCTGTAATTTCATAGCAGAA
CCTGGGTAAAGTTAACCTAAACCGTTAACATCAGTCATGATGAAATGTGAAAAAATACCTTCATATACTTTATTTA
GCAATAAGATAGGCTGACATAGTATCTCATAAATTAATATTGGAGGAACTGATGGA.
In the present invention, inventor shows lncRNA ENST00000490593.1 and pyemic hair by experimental verification
Raw related, the expression quantity in sepsis patient significantly raises, can be early with auxiliary judgment pyemia by detecting the lncRNA
Phase occurs and Later development, provides help for clinical diagnosis.The specifying information of LncRNA ENST00000490593.1 records
In website Ensembl Genome Browser (http://grch37.ensembl.org/Homo_sapiens/
Transcript/Summary? db=core；G=ENSG00000257335；R=7:141738346-141740241；T=
It ENST00000490593), is MGAM genetic transcription.
Second aspect, the present invention provide a kind of isolated polynucleotides, and the polynucleotides can be transcribed by people's cell
LncRNA described in first aspect.
In the present invention, a kind of isolated polynucleotides are provided, the polynucleotides can be transcribed into first aspect by people's cell
The structure of the lncRNA, the polynucleotides are typical but are Seq with not limiting toJust-X-SeqInstead, wherein SeqJustFor in people's cell
In be transcribed into the nucleotide sequence of the lncRNA, SeqInsteadWith SeqJustComplementary pairing, X SeqInsteadWith SeqJustBetween interval sequence
Column, the intervening sequence will not be with SeqInsteadWith SeqJustComplementation will not form self-complementary pairing, the isolated polynucleotides
Loop-stem structure is formed after being transferred to people's cell.
The third aspect, the present invention provide a kind of carrier, and the carrier contains lncRNA or second as described in relation to the first aspect
Polynucleotides described in aspect.
Fourth aspect, the present invention provide polynucleotides described in a kind of lncRNA as described in relation to the first aspect, second aspect
Or carrier described in the third aspect is in drug, chip, kit or the high-flux sequence platform of the screening of preparation pyemia or diagnosis
In application.
In the present invention, according to lncRNA ENST00000490593.1 and pyemic positive correlation, it can be applied to
In the product of various diagnosis of sepsis diseases, for example, drug, chip, kit or high-flux sequence platform.
Preferably, the application is to detect the expression of lncRNA described in first aspect in sample.
In the present invention, the expression quantity by detecting lncRNA ENST00000490593.1 judges whether corresponding sample suffers from
Pyemia determines result for the positive if expression quantity significantly raises.Detect the table of the lncRNA ENST00000490593.1
Method up to amount for example can be the methods of high-flux sequence, qPCR.
Preferably, the detection sample is blood.
5th aspect, the present invention provide a kind of lncRNA chip, and the lncRNA chip includes solid phase carrier and orderly
The oligonucleotide probe being fixed on the solid phase carrier, the oligonucleotide probe specifically correspond in first aspect
Sequence some or all of shown in SEQ ID NO.1.
In the present invention, according to the nucleotide sequence of lncRNA ENST00000490593.1, designs suitable specificity and visit
Needle is fixed on solid phase carrier, forms oligonucleotide arrays.The common material in genetic chip field can be used in the solid phase carrier
Material, such as can be nylon membrane, slide or plastic sheet etc..The preparation of chip can be used biochip known in the art and routinely grasp
Make method, the present invention does not do particular determination.
6th aspect, the present invention provide a kind of kit, and the kit includes for lncRNA as described in relation to the first aspect
Specific primer pair or probe.
Preferably, the sequence of the specific primer pair is as shown in SEQ ID NO.2-3.
SEQ ID NO.2:AAACATCCATCTACATGCTGAG.
SEQ ID NO.3:CTTTACCCAGGTTCTGCTATGA.
Preferably, the specific primer is visited to suitable for SYBR Green, Taqman probe, molecular beacon, double cross
The detection of needle or combined probe technology.
Compared with prior art, the invention has the following beneficial effects:
(1) present invention provides the lncRNA ENST00000490593.1 purposes for being used for sepsis markers, passes through gene
Chip and qPCR double verification, it was demonstrated that the lncRNA has transcriptional differences in sepsis patient into the cell；
(2) present invention provides a kind of lncRNA ENST00000490593.1 answering in the product for preparing diagnosis of sepsis disease
With, it lays the foundation for the research of pathogenesis of sepsis mechanism, while adjuvant clinical diagnoses, promotion patient's prognosis；
(3) present invention provides a kind of genetic chip, can carry out high-throughput detection lncRNA ENST00000490593.1's
Express spectra further enriches the research of pathogenesis of sepsis mechanism.
Detailed description of the invention
Fig. 1 is genechip detection result in the embodiment of the present invention 2；
Fig. 2 is qPCR testing result in the embodiment of the present invention 3.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below by way of specific embodiment come into
One step illustrates technical solution of the present invention, but the present invention is not limited in scope of embodiments.
1 sample rna of embodiment extracts
1, sample collection
Principles in Informed Consent is followed, and under the premise of Ethics Committee is agreed to, collects 12 healthy human bloods and 10
Sepsis patient blood.
2, prepared by RNA sample
It separates monocyte (MNCs) and extracts total serum IgE using RNAiso (TaKaRa, Dalian, China) kit afterwards.
Specifically: 1ml RNA extracting solution is added in cell, and 200ul chloroform (being equivalent to Trizol1/5 volume) is added in every pipe
Concussion mixes 15s, is stored at room temperature 5min, and 4 DEG C of 12000rpm are centrifuged 15min.Visible liquid in pipe is divided into 3 layers after centrifugation, and RNA is complete
Portion is dissolved in upper strata aqueous phase.Upper strata aqueous phase is carefully drawn into new 1.5mlEP pipe with 1ml liquid-transfering gun, adds equivalent isopropanol mixed
It is even, 10min is stood on ice, and 4 DEG C of 12000r/min are centrifuged 10min again, abandon supernatant.The ethyl alcohol of 1ml75% is added in every pipe, mildly
Concussion, 4 DEG C of 8000r/min are centrifuged 5min, as far as possible abandoning supernatant.EP pipe is upside down in room temperature on blotting paper to dry, it is seen that heavy in pipe
Forming sediment, it is transparent to be switched to by white, and DEPC water 10-20ul is added, is allowed to sufficiently dissolve, Total RNAs extraction finishes.
3, RNA sample quality inspection
Sample total serum IgE is quantitative using NanoDrop ND-2000 (Thermo Scientific) and through Agilent
Bioanalyzer 2100 (Agilent Technologies) detects RNA integrality.
2 genechip detection of embodiment
1, the reverse transcription and label of sample rna
After RNA quality inspection is qualified in embodiment 1, RNA reverse transcription further synthesizes cRNA at double-strand cDNA, then right
CRNA carries out the second wheel reverse transcription and synthesizes cDNA, fragmentation and and biotin labeling.
2, hybridize
Genetic chip uses Ou Yi biology-Affymetrix Human oelncRNA Array, by chip operation instructions
The step of hybridized, after eluting and dyeing using Affymetrix Scanner 3000 (Affymetrix) scanning obtain original
Beginning image.
3, data processing
Original image using Affymetrix GeneChip Command Console software (version4.0,
Affymetrix) processing extract initial data, followed by Expression Console software (version1.3.1,
Affymetrix the standardization of gene level and exon level, standardized algorithm RMA) are carried out to chip.Then it carries out
Gene expression analysis, analyzing the software used is Genespring software (version 13.1, Agilent
Technologies).Differential gene is screened using the T p value examined and fold change value, the standard of screening be up-regulation or
Lower fold change value >=value≤0.05 2.0 and P.
Genechip detection result is as shown in Figure 1, it is found that expression of the ENST00000490593.1 in sepsis patient
Level is significantly higher than Healthy People.
Embodiment 3qPCR detection
1, RNA reverse transcription
The OD260 of sample is measured by UV spectrophotometer measuring RNA concentration using 1 gained RNA sample of embodiment
The integrity degree and purity of RNA are determined with the ratio of OD260/OD280 with OD280., and according to the value of OD260, calculates rna content,
And suitably dilution adjustment total rna concentration about 1ug/ul.It then carries out reverse transcription and obtains cDNA, use oligo-Dt and mouse mastadenoma
Viral reverse transcriptase carries out RNA reverse transcription, Fermentas reverse transcription reagent box (Promega company), Oligo-dT (Jin Weizhi
Company)；
1) A system is established, system A is added in 200ul centrifuge tube and is mixed, the centrifugation of palm centrifuge is put to grads PCR instrument
In, 70 DEG C of incubation 5min are set, are transferred to 4 DEG C of 10min, the composition of system A is shown in Table 1；
Table 1
| Component | Volume |
| Oligo-dT | 8ul |
| DEPC water | 2ul |
| Total serum IgE | 2ul |
| Total volume | 12ul |
2) B system is established, system B is added in system A, mixes well, is reentered into after centrifugation in grads PCR instrument, if
Fixed 37 DEG C of incubations 60min, 95 DEG C of 5min switch to 4 DEG C and are shown in Table 2 to get the composition to cDNA, B system.
Table 2
| Component | Volume |
| RNA reverse transcription buffer | 5ul |
| DNTP mixture | 1.3ul |
| DEPC water | 5ul |
| RNAsin ribonuclease inhibitor | 0.7ul |
| RNA reverse transcriptase | 1ul |
| Total volume | 13ul |
2, qPCR reacts
QPCR experiment is carried out using SYBR Green master mix after design of primers, is made using house-keeping gene β-actin
For reference gene, primer particular sequence is as shown in SEQ ID NO.2-5.
The primer sequence of ENST00000490593.1 are as follows:
Forward primer (SEQ ID NO.2): 5 '-AAACATCCATCTACATGCTGAG-3 '；
Reverse primer (SEQ ID NO.3): 5 '-CTTTACCCAGGTTCTGCTATGA-3 '
The primer sequence of house-keeping gene β-actin are as follows:
Forward primer (SEQ ID NO.4): 5 '-CATGTACGTTGCTATCCAGGC-3 '；
Reverse primer (SEQ ID NO.5): 5 '-CTCCTTAATGTCACGCACGAT-3 '
Response procedures: reaction system is configured according to table 3, is loaded by above-mentioned reaction system, each sample does three multiple holes, so
Sample is placed in fluorescent quantitation instrument afterwards, is reacted according to the program set, reaction condition are as follows: 95 DEG C, 10min；95℃,
15s, 60 DEG C, 15s, 72 DEG C, 30s, 72 DEG C collection fluorescence, totally 40 recycle；Corresponding solubility curve is done after circulation terminates.With
LightCyler 480 included software carries out real-time data capture and quantitative analysis.
3 reaction system of table
| Component | Volume |
| SYBR Green master Mix | 10ul |
| Forward primer | 1ul |
| Reverse primer | 1ul |
| Deionized water | 7ul |
| cDNA | 1ul |
| Total volume | 20ul |
3, statistical analysis
Gene expression dose is standardized with β-actin, uses 2-ΔΔCtThe relative expression quantity of method calculating gene.Make
Gene expression amount between pyemia group and healthy control group is compared with Mann-Whitney U test.Statistical analysis makes
With GraphPad Prism software (GraphPad Software Inc.).Bilateral P value thinks less than 0.05 with statistically significant
Property, as a result see Fig. 2.
As shown in Figure 2, expression of the ENST00000490593.1 in sepsis patient is significantly higher than Healthy People, poor
It is different that there is statistical significance (P < 0.01), it is consistent with chip results.
The assembling of 4 kit of embodiment
RNA extracting solution: phenol, guanidinium isothiocyanate, beta -mercaptoethanol, 8-hydroxyquinoline；
RNA reverse transcriptase: moloney murine leukemia virus reverse transcriptase (M-MLV)；
Forward primer (SEQ ID NO.2): 5 '-AAACATCCATCTACATGCTGAG-3 '；
Reverse primer (SEQ ID NO.3): 5 '-CTTTACCCAGGTTCTGCTATGA-3 '
By RNA extracting solution, RNA reverse transcriptase, RNA reverse transcription buffer, Oligo-dT, SYBR Green master
Mix, primer (forward and reverse), deionized water, positive control, negative control and specification are packed into kit.
To sum up, the present invention provides a kind of lncRNA for sepsis markers, and finds lncRNA for the first time
ENST00000490593.1 and pyemic relationship, enrich the research of pathogenesis of sepsis mechanism, provide centainly for clinical diagnosis
Foundation.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>Fang Fang
<120>a kind of lncRNA and its application for sepsis markers
<130> 2019
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1896
<212> DNA
<213>artificial synthesized sequence
<400> 1
cagcacttgg gatgtgcacc aacagttctt atgggggccc ggcctcctca tcactccagt 60
tctggatgaa gtaagtgttc ccacagagat acactagaga tctctgcatc tgtatctgct 120
gccctgcaaa ctcctctctg cttcttattc caaactcact tctgacatct gtgactgagt 180
cagccttgaa gagagtgtgc taggttttga gaagctagac atctggtaac tacacatttt 240
tctttctaga atttcttttt tctcttgatc aagttcccaa ccagtttaga agcatctact 300
ggaaactcta gagttacaag ttatgttagg ttgttcttgc attgctatac agaaattcct 360
gacactgagt gatttacaaa gaaaagagat ttcattggct cacagttctg aaagctttac 420
aagaagcatg atgctggtat ctgtgcagct tctagggagg cctcaggaag cttacagtca 480
tggaggaagg tgaagaggag cagcgatgtc atatagcaaa agcaggaaca agacggggtg 540
gggggagttg ccacacactt tttttttttt ttttttttga gatggagtct cactctgtca 600
cccaggctgg agtgcagtgg cacaatccca gctcactgca acctccaact cctgggttca 660
catgattctc ctgcctcagc ctcctgagta gctggggtta caggcacctg ccaccaagct 720
cggctaattt tgtatttttt gtagagacgg ggtttcatgt tagccaggct ggtctcaaac 780
tgctgacctc agatgatcca cctgcctcag cctccaaagt gctgagatta caggtgtgag 840
tcaccgcgct tggccgccac acacttttaa acaaccaaat cttacaagaa ttcactcact 900
atcaccagga cagaaccaag ggaatggtgt aaaccattca tgagaaatct gccctcatca 960
tccagtcagc tccctccagg ccccacctcc aacactgggg attacatttc aacatgagat 1020
ttgggcagac aaacatccaa actatatcaa gagtggtcct atttgttgtt ggattctcat 1080
agcagatcct gacttgctcc atgacgtaga aacaggatgg agcagtgacc caacactcac 1140
tctaaagtca ggctgatcgg gatgtgattg ttgtctctgc cactttgagc ctgtgtgatc 1200
aggggtgact attgtgctgg cttctcatct gcagaggggt aatactacta ctactaaaaa 1260
taatggtacc caccatttgg gaatgttggg aaaattaaat gaggtaatca tgtaaagctg 1320
gaatgtttgt caccacacat gttttattat tattttatga ttattattat tcctaatgtg 1380
gctacgtttg ggatttctct ctgggttgcc aagttacagt ttctattatt tgtattagag 1440
gagaagcaga agaaaggcat aatagcaggg cattcctgcc ctctctgtgt atgattaaga 1500
aattccctag agaataaaaa gaaatccatt ttatttccct caggggacac tacatttttt 1560
taatttcagg gtgcagagaa agtgatggca tatgtgcctg atgctgtctg gtatgactac 1620
gagactgtaa gtagctttga cttttcttct actccttaag actgtagctg cagctgcata 1680
gacaagctac ctttctggag agagaaacat ccatctacat gctgaggagt agtttttagt 1740
gttttctgta atttcatagc agaacctggg taaagttaac ctaaaccgtt aacatcagtc 1800
atgatgaaat gtgaaaaaat accttcatat actttattta gcaataagat aggctgacat 1860
agtatctcat aaattaatat tggaggaact gatgga 1896
<210> 2
<211> 22
<212> DNA
<213>artificial synthesized sequence
<400> 2
aaacatccat ctacatgctg ag 22
<210> 3
<211> 22
<212> DNA
<213>artificial synthesized sequence
<400> 3
ctttacccag gttctgctat ga 22
<210> 4
<211> 21
<212> DNA
<213>artificial synthesized sequence
<400> 4
catgtacgtt gctatccagg c 21
<210> 5
<211> 21
<212> DNA
<213>artificial synthesized sequence
<400> 5
ctccttaatg tcacgcacga t 21
Claims (10)
1. a kind of lncRNA for sepsis markers, which is characterized in that the lncRNA is lncRNA
ENST00000490593.1, nucleotide sequence is as shown in SEQ ID NO.1.
2. a kind of isolated polynucleotides, which is characterized in that the polynucleotides can be transcribed into claim 1 institute by people's cell
The lncRNA stated.
3. a kind of carrier, which is characterized in that the carrier contains described in lncRNA as described in claim 1 or claim 2
Polynucleotides.
4. a kind of lncRNA as described in claim 1, polynucleotides as claimed in claim 2 or load as claimed in claim 3
Application of the body in the drug of the screening of preparation pyemia or diagnosis, chip, kit or high-flux sequence platform.
5. application according to claim 4, which is characterized in that the application is described in claim 1 in detection sample
The expression of lncRNA.
6. application according to claim 5, which is characterized in that the detection sample is blood.
7. a kind of lncRNA chip, which is characterized in that the lncRNA chip includes solid phase carrier and is orderly fixed on described
Oligonucleotide probe on solid phase carrier, the oligonucleotide probe specifically correspond to SEQ ID NO.1 in claim 1
Some or all of shown sequence.
8. a kind of kit, which is characterized in that the kit includes drawing for the specificity of lncRNA as described in claim 1
Object pair or probe.
9. kit according to claim 8, which is characterized in that the sequence of the specific primer pair such as SEQ ID
Shown in NO.2-3.
10. kit according to claim 8, which is characterized in that the specific primer to be suitable for SYBR Green,
The detection of Taqman probe, molecular beacon, double cross probe or combined probe technology.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226491A (en) * | 2020-10-15 | 2021-01-15 | 赣南医学院 | Screening method of biomarker for evaluating sepsis |
| CN113501862A (en) * | 2021-05-26 | 2021-10-15 | 上海市第一人民医院 | Polypeptide and application thereof in preparation of immunoregulation medicament |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2971168A1 (en) * | 2014-12-18 | 2016-06-23 | Csir | Immunomodulation by controlling elr+ proinflammatory chemokine levels with the long non-coding rna umlilo |
| CN109022572A (en) * | 2018-09-14 | 2018-12-18 | 北京泱深生物信息技术有限公司 | LOC101927627 and its application as sepsis diagnosis marker |
| CN109112212A (en) * | 2018-09-14 | 2019-01-01 | 北京泱深生物信息技术有限公司 | sepsis diagnosis marker |
-
2019
- 2019-04-24 CN CN201910335045.7A patent/CN109988830A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2971168A1 (en) * | 2014-12-18 | 2016-06-23 | Csir | Immunomodulation by controlling elr+ proinflammatory chemokine levels with the long non-coding rna umlilo |
| CN109022572A (en) * | 2018-09-14 | 2018-12-18 | 北京泱深生物信息技术有限公司 | LOC101927627 and its application as sepsis diagnosis marker |
| CN109112212A (en) * | 2018-09-14 | 2019-01-01 | 北京泱深生物信息技术有限公司 | sepsis diagnosis marker |
Non-Patent Citations (1)
| Title |
|---|
| 戴巍等: "长链非编码RNA在脓毒症肺损伤中作用的研究进展", 江西医药 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226491A (en) * | 2020-10-15 | 2021-01-15 | 赣南医学院 | Screening method of biomarker for evaluating sepsis |
| CN113501862A (en) * | 2021-05-26 | 2021-10-15 | 上海市第一人民医院 | Polypeptide and application thereof in preparation of immunoregulation medicament |
| CN113501862B (en) * | 2021-05-26 | 2022-09-13 | 上海市第一人民医院 | Polypeptide and application thereof in preparation of immunoregulation medicament |
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