CN106086017A - ENST00000509938.1 and preparation or diagnostic agent or medicine or test kit and application - Google Patents
ENST00000509938.1 and preparation or diagnostic agent or medicine or test kit and application Download PDFInfo
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Abstract
The present invention relates to gene engineering technology field, be a kind of long-chain non-coding RNA ENST00000509938.1 and test kit thereof and application.Long-chain non-coding RNA and preparation thereof or diagnostic agent or medicine or test kit and application, this long-chain non-coding RNA sequence has the sequence shown in SEQ ID NO:1.Compared with the blood content of Healthy People, long-chain non-coding RNA ENST00000509938.1 significantly high expression in patients of acute myocardial infarction blood.Long-chain non-coding ENST00000509938.1 of the present invention will enrich the research of acute myocardial infarction mechanism further, and also early diagnosis and Prognosis scoveillance for acute myocardial infarction provide new blood molecules mark and therapy target.
Description
Technical field
The present invention relates to gene engineering technology field, be a kind of LncRNA ENST00000509938.1 and preparation or diagnosis
Agent or medicine or test kit and application i.e. ENST00000509938.1 and preparation or diagnostic agent or medicine or test kit and application.
Background technology
Acute myocardial infarction (acute myocardial infarction, AMI) is as coronary heart disease
Type the most serious in (Coronary heart disease, CHD), its morbidity is anxious, case fatality rate is high, forms people's health
Serious threat.It is raw that the diagnosis of acute myocardial infarction at present depends on clinical manifestation, Electrocardiographic dynamic change and serum more
Thing mark.Diagnosing acute myocardial infarction and preventing the most earlier, is always the important topic of our research.And with
The Human Genome Project and complete the development with biochip technology, the Disease-causing gene of many single-gene disorders is found, and should
Use in the middle of the Clinics and Practices of disease.And AMI exists as a kind of disease affected by multiple nature-nurture factor, hereditism
Effect in AMI morbidity is the clearest and the most definite.
Long-chain non-coding RNA (long non-coding RNAs, lncRNA) is that a class transcript length is more than 200nt
RNA molecule, the most not encoding proteins, transcribed by rna plymerase ii and formed through variable sheer, and transcriptional level is compiled less than protein
Code gene, had been considered as the most once " noise " of subgenomic transcription.It has proven convenient that lncRNA epigenetics, transcriptional control, turn
After record, the multiple aspect such as regulation and control controlling gene is expressed, and plays a significant role in various biological process, including lipid metabolism, moves
Pulse atherosclerosis formation, fetal development and the generation etc. of tumor,
Long-chain non-coding RNA (long noncoding RNA, lncRNA) is that a class can be sent out during various biological
Wave the macromole ncRNA of regulating and controlling effect.It is widely distributed, and length is typically greater than 200 bases, owing to lacking effective open reading
Frame (ORF) and without or seldom have the ability of encoding proteins.As a uncharted field in molecular biology, lncRNA be with
The form of RNA plays the effect that controlling gene is expressed in the multiple aspects such as epigenetics, transcriptional control, post-transcriptional control.
As the important component part of mammalian transcription group, the function of lncRNA needs to further investigate at present.Originally lncRNA
It is considered as the by-product transcribed of RNA polymerase II, is subgenomic transcription " noise ", not there is biological function.But in recent years
Result of study show, lncRNA important function in cell normal physiological activity and participate in multiple disease and send out
Exhibition.LncRNA can be reconstructed by Genomic Imprinting, chromatin, the mechanism such as regulation and control, mRNA degraded and translational control of shearing implement it
Function.
Summary of the invention
The invention provides a kind of long-chain non-coding RNA ENST00000509938.1 and preparation thereof or diagnostic agent or medicine
Or test kit and application, overcoming the deficiency of above-mentioned prior art, it can effectively solve acute myocardial infarction as one by many
The disease of kind of nature-nurture factor impact, but the problem that effect that hereditism is in acute myocardial infarction is the clearest and the most definite,
Present invention discover that it is expressed or dysfunction and the generation of human cardiovascular disease and develop closely related, long-chain non-coding RNA
ENST00000509938.1 is expected to become cardiovascular disease diagnosis, the mark of prediction prognosis, the most also controlling for cardiovascular diseases
Treat and provide new target spot.
One of technical scheme is realized by following measures: a kind of at patients of acute myocardial infarction blood
The long-chain non-coding RNA ENST00000509938.1 of middle high expressed, this long-chain non-coding RNA ENST00000509938.1's
Sequence is as shown in SEQ ID NO:1.
Further optimization and/or improvements to foregoing invention technical scheme one of are presented herein below:
Above-mentioned long-chain non-coding lncRNA ENST00000509938.1 is the total length of ENST00000509938:1
The two of technical scheme are realized by following measures: according to technology in a kind of vitro detection blood
The preparation of the long-chain non-coding RNA ENST00000509938.1 that one of scheme is described or diagnostic agent or medicine or test kit, should
Preparation or diagnostic agent or medicine or test kit include specific primer to, standard DNA template, PCR reactant liquor, wherein, specificity
Primer is to including forward primer and downstream primer, and the nucleotides sequence of forward primer is classified as SEQ ID No:2, the nucleoside of downstream primer
Acid sequence is SEQ ID No:3.
Be presented herein below to foregoing invention technical scheme two further optimization and/or improvements:
This test kit above-mentioned is fluorescent quantificationally PCR detecting kit.
Above-mentioned specific primer is to for SYBR Green, Taqman probe, molecular beacon, double cross probe, compound spy
The detection of pin.
Above-mentioned PCR reactant liquor is fluorescence quantitative PCR reaction solution.
Above-mentioned fluorescence quantitative PCR reaction solution includes dNTP, Mg2+, Taq enzyme.
Above-mentioned also include fluorescent dye.
The three of technical scheme are realized by following measures: a kind of detection is according to one of technical scheme institute
The method of the long-chain non-coding RNA ENST00000509938.1 stated, is carried out: the first step in the steps below, extracts blood total
RNA；Second step, prepares cDNA；3rd step, quantitative amplification lncRNA ENST00000509938.1.
The four of technical scheme are realized by following measures: a kind of according to one of technical scheme Suo Shu
Long-chain non-coding RNA ENST00000509938.1 is used for detecting the test kit of acute myocardial infarction or is used as detection anxious in preparation
The application of the novel targets of property myocardial infarction medicine.
Long-chain non-coding RNA ENST00000509938.1 in the present invention is notable in patients of acute myocardial infarction blood
High expressed, and it is further characterized by long-chain non-coding RNA ENST00000509938.1 in urgency in the fluorescent quantitation of sample is tested
Being significantly higher than Healthy People in property myocardial infarction patient blood, long-chain non-coding ENST00000509938.1 of the present invention will enter one
The research of step abundant acute myocardial infarction mechanism, also early diagnosis and Prognosis scoveillance for acute myocardial infarction provide newly
Blood molecules mark and therapy target.
Accompanying drawing explanation
Accompanying drawing 1 is that in the present invention, long-chain non-coding RNA chip of expression spectrum detects ENST00000509938.1 in normal healthy controls
Group and the detected signal value in acute myocardial infarction group.
Accompanying drawing 2 is a pair spy of the sequential design in the present invention for long-chain non-coding RNA ENST00000509938.1
The effect of row agarose gel electrophoresis test primer after specific primer PCR amplification.
Accompanying drawing 3 is that in the present invention, qRT-PCR detects ENST00000509938.1 at normal healthy controls group and acute myocardial infarction
Relative expression quantity in group
Detailed description of the invention
The present invention is not limited by following embodiment, can determine specifically according to technical scheme and practical situation
Embodiment.
In the present invention, for the ease of describing, the description of the relative position relation of each parts is all according to Figure of description 1
Butut mode be described, such as: the position relationship of upper and lower, left and right etc. is based on the Butut direction of Figure of description to be come
Determine.
The present invention utilizes long-chain non-coding chip of expression spectrum technology, by variation analysis, screens one at Acute myocardial
The lncRNA ENST00000509938.1 of significantly high expression in Infarction Patients blood, its transcript regions is positioned at No. 4 chromosomes, rises
Beginning position is 113,567,878-113,569,855, and total length 1978bp.Compared with the blood content of Healthy People, lncRNA
ENST00000509938.1 is significantly high expression in patients of acute myocardial infarction blood, and in the fluorescent quantitation of sample is tested
It is further characterized by lncRNA ENST00000509938.1 and is significantly higher than Healthy People in patients of acute myocardial infarction blood.Cause
This, long-chain non-coding lncRNA ENST00000509938.1 will enrich the research of acute myocardial infarction mechanism further,
Also early diagnosis and Prognosis scoveillance for acute myocardial infarction provide new blood molecules mark and therapy target.
The patients of acute myocardial infarction of present invention offer and each 3 of the blood preparation of Healthy People, totally 6 example.By ABI company
Trizol reagent (article No. 15596-026) needed for step extract after total serum IgE, use Beijing Bo Ao biological engineering company limited
Chip product (Agilent human lncRNA+mRNA Array V4.0) detects, and filters out one and obstructs at Acute myocardial
The lncRNA ENST00000509938.1 of significantly high expression in dead blood samples of patients, its nucleotide sequence is as shown in SEQ ID No.1.
Later stage through sample is carried out quantitative fluorescent PCR checking, finds in 6 samples lncRNA in patients of acute myocardial infarction blood
The expression of ENST00000509938.1 is significantly higher than the expression of lncRNA ENST00000509938.1 in healthy human blood.
LncRNA ENST00000509938.1 is as the novel targets of acute myocardial infarction, for clinical treatment and the medicine of acute myocardial infarction
Thing exploitation is provided fundamental basis.
Embodiment 1, a kind of long-chain non-coding RNA of high expressed in patients of acute myocardial infarction blood
ENST00000509938.1, the sequence of this long-chain non-coding RNA ENST00000509938.1 is as shown in SEQ ID NO:1.
Embodiment 2, as the optimization of embodiment 1, long-chain non-coding lncRNA ENST00000509938.1 is
The total length of ENST00000509938:1
Embodiment 3, according to the long-chain non-coding RNA described in embodiment 1 or embodiment 2 in a kind of vitro detection blood
The preparation of ENST00000509938.1 or diagnostic agent or medicine or test kit, said preparation or diagnostic agent or medicine or test kit bag
Include specific primer to, standard DNA template, PCR reactant liquor, wherein, specific primer to including forward primer and downstream primer,
The nucleotides sequence of forward primer is classified as SEQ ID No:2, and the nucleotides sequence of downstream primer is classified as SEQ ID No:3.
Embodiment 4, as the optimization of embodiment 3, this test kit is fluorescent quantificationally PCR detecting kit.
Embodiment 5, as embodiment 3 or the optimization of embodiment 4, specific primer is to for SYBR Green, Taqman
Probe, molecular beacon, double cross probe, the detection of combined probe.
Embodiment 6, as embodiment 3, embodiment 4 and the optimization of embodiment 5, PCR reactant liquor is quantitative fluorescent PCR reaction
Liquid.
Embodiment 7, as the optimization of embodiment 6, fluorescence quantitative PCR reaction solution includes dNTP, Mg2+, Taq enzyme.
Embodiment 8, as embodiment 3, embodiment 4, embodiment 5, embodiment 6 and the optimization of embodiment 7, this test kit is also
Including fluorescent dye.
The using method of the dye class PCR kit for fluorescence quantitative of this vitro detection acute myocardial infarction, quantitative fluorescent PCR
System:
Forward primer, downstream primer each 0.25ul (10uM)；DNA profiling cDNA 0.5ul；Power SYBR Green
PCR Master (2 ×) 5ul, adds Nuclease-Free Water4ul, total capacity 10ul.Quantitative fluorescent PCR program: the first step
95 DEG C 10 minutes；2nd step, 95 DEG C of 15S, 60 DEG C 1 minute；3rd step, 95 DEG C of 15S, 60 DEG C 1 minute, 95 DEG C of 15S, totally 40 are followed
Ring.
Embodiment 9, a kind of detection is according to the long-chain non-coding RNA described in embodiment 1 and embodiment 2
The method of ENST00000509938.1, is carried out: the first step in the steps below, extracts blood total serum IgE；Second step, prepares cDNA；
3rd step, quantitative amplification lncRNA ENST00000509938.1.
Described method specifically includes following steps:
One, blood total serum IgE is extracted: according to reagent and step needed for the Trizol reagent (article No. 15596-026) of ABI company
Extract total serum IgE, more fixed with 7300real time PCR system nucleic acid quantification instrument quantitatively (Applied Biosystems AB)
Amount extracted purity and concentration.
Two, sample cDNA is prepared: use Beijing Tian Gen biochemical technology company FastQuant cDNA the first chain synthetic agent
The box test kit (article No. KR106) the total serum IgE reverse transcription synthesis cDNA to extracting.
1, template ribonucleic acid is thawed on ice；5×gDNA Buffer、FQ-RT Primer Mix、10×Fast RT
Buffer、RNase-Free ddH2O thaws in room temperature (15-25 DEG C), is immediately placed on ice after defrosting.Use front molten by every kind
Liquid vortex oscillation mixes, and brief centrifugation remains in the liquid of tube wall to collect.
2, opening secondary structure, reaction system and condition are as shown in table 1；
The removal system of the genomic DNA of said components is prepared mixed liquor, thoroughly mixes.Brief centrifugation, is placed in 42
DEG C, hatch 3min.It is subsequently placed in and places on ice.
3, reverse transcription reaction, reaction system and condition are as shown in table 2；
By the Mix in reverse transcription reaction, it is added in the reactant liquor of gDNA removal step, fully mixes.42 DEG C, hatch
15min.95 DEG C, being put on ice after hatching 3min, the cDNA obtained can be used for subsequent experimental, or cryopreservation.
Three, amplification lncRNA ENST00000509938.1: use the Power SYBR Green PCR of ABI company
Master Mix fluorescence quantitative kit (article No. 4367659), carries out quantitative fluorescent PCR expansion with the cDNA of reverse transcription for template
Increase.
Quantitative fluorescent PCR system is as shown in table 3；
Quantitative fluorescent PCR program: the first step 95 DEG C 10 minutes；2nd step, 95 DEG C of 15S, 60 DEG C 1 minute；3rd step, 95 DEG C
15S, 60 DEG C 1 minute, 95 DEG C of 15S, totally 40 circulations.
Embodiment 10, a kind of according to the long-chain non-coding RNA described in embodiment 1 and embodiment 2
ENST00000509938.1 is used for detecting the test kit of acute myocardial infarction or being used as detection acute myocardial infarction medicine in preparation
The application of novel targets.
For the method detecting acute myocardial infarction, said method comprising the steps of:
The first step, extracts blood total serum IgE；
Second step, prepares sample cDNA；
3rd step, expands lncRNA ENST00000509938.1, and judges according to relative quantification result.
Embodiment 11: patients of acute myocardial infarction and the lncRNA chip expression analysis of healthy human blood
One, material and method
1. material
Blood sample comes from patients of acute myocardial infarction and each 3 of healthy human blood's sample.
2. method
(1) patients of acute myocardial infarction and the extraction of healthy human blood's sample total serum IgE: according to the rich limited public affairs of biotechnology difficult to understand
" for the preparation process of blood sample of gene microarray analysis " that department provides extracts patients of acute myocardial infarction and healthy human blood
Liquid sample total serum IgE.Specific experiment material includes: lymphocyte separation medium (TBD biotech development center, Tianjin)；Sterile physiological
Saline (common 0.9% normal saline sterilizing)；Trizol reagent (Invitrogen company).
(2)The detection of lncRNA+mRNA chip of expression spectrum is initial with the total serum IgE of detected sample, carries out external expansion
Increasing and fluorescent labeling, labeling process usesBiochip common tags test kit.Mainly comprise the steps:
1) reverse transcription synthesizes the first chain cDNA: initiate with Total RNA, the T7Oligo (dT) containing T7 promoter sequence
Primer and T7-random primer, both can be in conjunction with the mRNA of band poly (A), it is also possible to combine in addition to rRNA other without
The RNA of poly (A), uses the first chain Enzyme Mix to synthesize the first chain cDNA.
2) the second chain DNA is synthesized: with the second chain Enzyme Mix, the RNA chain in DNA-RNA heterozygote is converted into second
Chain cDNA, synthetic dsdna.
3) in vitro transcription synthesis cRNA: with the second chain cDNA as template, utilizes T7Enzyme Mix to synthesize cRNA.
4) cRNA purification: use RNA purification column purification cRNA, removes the reagent such as the salt in reaction, enzyme, and carries out cRNA
Quantitatively, Quality Control.
5) reverse transcription: with cRNA as template, Random Primer is primer, and CbcScript II enzyme carries out reverse transcription.Pure
The cDNA that change recovery reverse transcription obtains is the most quantitatively.
6) labelling: with the cDNA product of reverse transcription as template, Random Primer is primer, uses Klenow Fragment
Enzymatic synthesis cDNA complementary strand also mixes the dNTP (Cy3-dCTP, Cy5-dCTP) with fluorophor, and purification quantitative mark are produced
Thing.Chip hybridization is i.e. can be used for fluorophor DNA.
7) chip hybridization and cleaning: take 1 μ LcDNA and be dissolved in hybridization solution, 45 DEG C of hybridized overnight.Take out chip at rich Slide difficult to understand
Washer8 chip is washed dry instrument and is carried out, and cleaning procedure is as follows: washing liquid I:0.2%SDS, 2 × SSC, and 42 DEG C of 120S clean 2 times.
Washing liquid II:0.2%SDS, 2 × SSC, 42 DEG C of 80S clean 3 times.
After cleaning procedure completes, centrifuge dripping, to be scanned.
8) chip scanning, data analysis, differential gene screens: use Agilent chip scanner (G2565CA) to cleaning
After chip be scanned, obtain hybridize picture.Use Agilent Feature Extraction (v10.7) software to hybridization
Picture is analyzed and extracts data.Then use Agilent GeneSpring software that data are normalized and difference is divided
Analysis, and carry out differential gene screening with GeneSpring GX software.
Two, result
Long-chain non-coding chip of expression spectrum detection ENST00000509938.1 is in normal healthy controls group and acute myocardial infarction group
In detected signal value as shown in Figure 1.Chip examination finds a plurality of up-regulated and the lncRNAs of down-regulated expression.Wherein
LncRNA ENST00000509938.1 demonstrates and expresses notable rise in patients of acute myocardial infarction, and Fc value is 2.818 times,
Wherein, upper from figure, P is less than 0.05, has statistical significance.In view of it may exist spy in patients of acute myocardial infarction
The opposite sex is expressed, and the present invention uses the sample of chip detection to carry out the repeated authentication of index by following example.
Embodiment 12:qRT-PCR repeated authentication lncRNA ENST00000509938.1 in patients of acute myocardial infarction and
Differential expression in Healthy People
One, experiment material
Choose the sample of chip testing, the differential expression of lncRNA ENST00000509938.1 is carried out qRT-PCR and tests
Card.
Two, experimental technique and result
1. primer specificity is identified
(1) design of specific primer: extract lncRNA ENST00000509938.1 from Ensemble data base and be correlated with
Transcript sequence, and drawn by the design of the design of primers instrument (Primer BLAST) of NCBI by the sequence according to transcript
Thing；
Primer sequence after design is as follows:
Forward primer: SEQ ID No:2
Downstream primer: SEQ ID No:3
(2) by patients of acute myocardial infarction and healthy human blood according to Trizol reagent (the article No. 15596-of ABI company
026) needed for, reagent and step extract total serum IgE, more quantitative with 7300 real time PCR system nucleic acid quantification instrument
(Applied Biosystems AB) quantitatively extracted purity and concentration.
Use Beijing Tian Gen biochemical technology company FastQuant cDNA the first chain synthetic agent box test kit (article No.
KR106) to the total serum IgE reverse transcription synthesis cDNA extracted.
Template ribonucleic acid is thawed by the first step on ice；5×gDNA Buffer、FQ-RT Primer Mix、10×Fast RT
Buffer、RNase-Free ddH2O thaws in room temperature (15-25 DEG C), is immediately placed on ice after defrosting.Use front molten by every kind
Liquid vortex oscillation mixes, and brief centrifugation remains in the liquid of tube wall to collect.
It is as shown in table 4 that second step opens secondary structure, reaction system and condition,
The removal system of the genomic DNA of said components is prepared mixed liquor, thoroughly mixes.Brief centrifugation, is placed in 42
DEG C, hatch 3min.It is subsequently placed in and places on ice.
3rd step reverse transcription reaction, reaction system and condition are as shown in table 5:
By the Mix in reverse transcription reaction, it is added in the reactant liquor of gDNA removal step, fully mixes.42 DEG C, hatch
15min.95 DEG C, being put on ice after hatching 3min, the cDNA obtained can be used for subsequent experimental, or cryopreservation.
3) amplification lncRNA ENST00000509938.1: use the Power SYBR Green PCR of ABI company
Master Mix fluorescence quantitative kit (article No. 4367659), carries out quantitative fluorescent PCR expansion with the cDNA of reverse transcription for template
Increase.
Quantitative fluorescent PCR system is as shown in table 6；
Quantitative fluorescent PCR program: the first step 95 DEG C 10 minutes；2nd step, 95 DEG C of 15S, 60 DEG C 1 minute；3rd step, 95 DEG C
15S, 60 DEG C 1 minute, 95 DEG C of 15S, totally 40 circulations.
Electrophoresis detection, selects 0120000Marker (Beijing CoWin Bioscience Co., Ltd., article No. CW0632) knot
The most as shown in Figure 2: amplified fragments size is identical with expection, amplified production only one of which band.This primer is to meeting above-mentioned standard.
The specific primer of upstream, its sequence is shown in sequence table SEQ ID No.2, and the specific primer in downstream, its sequence is shown in sequence table SEQ
ID No.2。
2. the preparation of standard DNA template
According to lncRNA ENST00000509938.1 base sequence (its nucleotide sequence such as sequence table SEQ ID No.1 institute
Show), entrust Shanghai raw work synthesis.
Sampling 2ul synthetic product, is connected to Puc-TTA cloning vehicle (Beijing CoWin Bioscience Co., Ltd., goods
Number CW0801), it is transformed into subsequently in DH5a competent cell.It is SEQ ID No.2 by sequence and SEQ ID No.3 special
Property primer screening positive colony, extract plasmid DNA, plasmid DNA is with 7300 real time PCR system nucleic acid quantification instrument calmly
Amount, and (standard DNA template concentration range is 10 as standard curve to do 10 times of serial dilutions2-106Copy/ul).
3. sensitivity experiments is by standard
Standard DNA template plasmid is diluted to 10 in proportion2、103、104、105、106Copy/ul, carries out quantitative fluorescent PCR
Detection sensitivity.Concentration limit is 102Copy/ul.
4. synthesis cRNA template
Take each 3 of the total serum IgE of above-mentioned acute myocardial infarction and Healthy People, use Beijing Tian Gen biochemical technology company
The FastQuant cDNA the first chain synthetic agent box test kit (article No. KR 106) the total serum IgE reverse transcription synthesis cDNA to extracting.
Template ribonucleic acid is thawed by the first step on ice；5×gDNA Buffer、FQ-RT Primer Mix、10×Fast RT
Buffer, RNase-Free ddH2O thaws in room temperature (15-25 DEG C), is immediately placed on ice after defrosting.Use front molten by every kind
Liquid vortex oscillation mixes, and brief centrifugation remains in the liquid of tube wall to collect.
It is as shown in table 7 that second step opens secondary structure, reaction system and condition:
The removal system of the genomic DNA of said components is prepared mixed liquor, thoroughly mixes.Brief centrifugation, is placed in 42
DEG C, hatch 3min.It is subsequently placed in and places on ice.
3rd step reverse transcription reaction, reaction system and condition are as shown in table 8:
By the Mix in reverse transcription reaction, it is added in the reactant liquor of gDNA removal step, fully mixes.42 DEG C, hatch
15min.95 DEG C, being put on ice after hatching 3min, the cDNA obtained can be used for subsequent experimental, or cryopreservation.
5. fluorescence quantitative PCR detection lncRNA ENST00000509938.1
Use the Power SYBR Green PCR Master Mix fluorescence quantitative kit (article No. of ABI company
4367659), fluorescent quantitative PCR is carried out with the cDNA of reverse transcription for template.
Quantitative fluorescent PCR system is as shown in table 9:
Quantitative fluorescent PCR program: the first step 95 DEG C 10 minutes；2nd step, 95 DEG C of 15S, 60 DEG C 1 minute；3rd step, 95 DEG C
15S, 60 DEG C 1 minute, 95 DEG C of 15S, totally 40 circulations.
QRT-PCR detection ENST00000509938.1 relative expression in normal healthy controls group with acute myocardial infarction group
Measure as shown in Figure 3, according to the relative quantification formula of qRT-PCR: 2-△△ct, calculate lncRNA respectively
ENST00000509938.1 expression in patients of acute myocardial infarction (AMI) and Healthy People (N), acute shown in result
Myocardial infarction patient (AMI) compares with the lncRNA ENST00000509938.1 expression of Healthy People, and they are 2 years old-△△ctValue is respectively
Being 2.1622,0.7299,1.0747, its meansigma methods is 1.6184.These results suggest that, lncRNA ENST00000509938.1
Universal high expressed in patients of acute myocardial infarction blood, this and foregoing lncRNA chip results
ENST00000509938.1 rise 2.818 times is consistent, and wherein, from figure, P is less than 0.05, has statistical significance.
Therefore, lncRNA ENST00000509938.1 can use as the new blood biomarker of Diagnosis of Acute Myocardial Infarction
Examination in acute myocardial infarction.
Above technical characteristic constitutes embodiments of the invention, and it has stronger adaptability and implementation result, can basis
It is actually needed the non-essential technical characteristic of increase and decrease, meets the demand of different situations.
Table 1
| Reagent | Consumption |
| 5×gDNA Buffer | 2ul |
| Total serum IgE | 1-2ug |
| DEPC water | Complement to 10uL |
Table 2
| Reagent | Consumption |
| 10×Fast RT Buffer | 2ul |
| RT Enzyme Mix | 1ul |
| FQ-RT Primer Mix | 2ul |
| RNase-Free ddH2O | Supply 10 μ l |
Table 3
| Reagent | Consumption |
| Power SYBR Green PCR Master(2×) | 5ul |
| CDNA sample | 0.5ul |
| Forward Primer(10μM) | 0.25ul |
| Reverse Primer(10μM) | 0.25ul |
| Nuclease-Free Water | 4ul |
| Total capacity | 10ul |
Table 4
| Reagent | Consumption |
| 5×gDNA Buffer | 2ul |
| Total serum IgE | 1-2ug |
| DEPC water | Complement to 10uL |
Table 5
| Reagent | Consumption |
| 10×Fast RT Buffer | 2ul |
| RT Enzyme Mix | 1ul |
| FQ-RT Primer Mix | 2ul |
| RNase-Free ddH2O | Supply 10 μ l |
Table 6
| Reagent | Consumption |
| Power SYBR Green PCR Master(2×) | 5ul |
| CDNA sample | 0.5ul |
| Forward Primer(10μM) | 0.25ul |
| Reverse Primer(10μM) | 0.25ul |
| Nuclease-Free Water | 4ul |
| Total capacity | 10ul |
Table 7
| Reagent | Consumption |
| 5×gDNA Buffer | 2ul |
| Total serum IgE | 1-2ug |
| DEPC water | Complement to 10uL |
Table 8
| Reagent | Consumption |
| 10×Fast RT Buffer | 2ul |
| RT Enzyme Mix | 1ul |
| FQ-RT Primer Mix | 2ul |
| RNase-Free ddH2O | Supply 10 μ l |
Table 9
| Reagent | Consumption |
| Power SYBR Green PCR Master(2×) | 5ul |
| CDNA sample | 0.5ul |
| Forward Primer(10μM) | 0.25ul |
| Reverse Primer(10μM) | 0.25ul |
| Nuclease-Free Water | 4ul |
| Total capacity | 10ul |
Sequence table
<110>No.1 Hospital Attached to Xinjiang Medical Univ.
<120>long-chain non-coding RNA ENST00000509938.1 is as the blood molecules mark of Diagnosis of Acute Myocardial Infarction
Detection and application
<130>
<160>
<170>
<210> 1
<211> 1978
<212> DNA
<213> Homo sapiens
<400> 1
1 cttcactgta aaatacttat aatctaaagg aatcgactca tatgtttata tttgctacaa
61 atggattttt ctaaattaca acaacataaa agccaattta atgtttttgt agagatatta
121 ttcttaagag ggtagaattg catgaattct gacctttggg gaagatcttg tatgataatg
181 gtttagatta aatattggcc aattctctca aattcttgtg tgagatattt agaagttaat
241 tacttaaaaa tctggtaaca ggtatattgt tttgttttat attctctatt taaaagaaac
301 tttgaatgca tttttaatta acctctatag atatatacaa gttgtacatg gtcctttcag
361 ccattttcct acattgtacc agaattatta gaacaaaact ggatatccca tgcaaaattt
421 aaaagttgga cccttacctt acaccatatg caaaacttaa aatggatcaa agacctaaat
481 gtaagagcta aagctatgaa actcttagaa gaacatgtag gagaaaaact ttcatgacat
541 tggatttgcc aacaattttt ttggatatga cacaaaagca caagcaacaa aagaaaaaat
601 agatacattg aacctcatca aaattaaaaa ccttttatgc atcaaaggac acaatcaaga
661 gagtaaaaat atgggctagg cacagtggct caggcctcta atcacagcac tttgggaggc
721 agaggtggga gaatcacttg aggtcaggag tttgagacca gcctgggaaa catagcaaga
781 ccctgtctct acaaaaagaa aaattaatga aaagataacc tatagaatag gagaaaatat
841 ttgcaaatta tacatctagt aaggggttaa tatccggaat atataaagaa cttctgtaac
901 ttaacaacaa caaaaaagca attaaaaaat gagtaaaagg cttgaataaa catttcttca
961 aagaagatat acaaatgacc aataagccca tagaaagaca ctcaacatta ctcatcatta
1021 gagaaataca catcaaacca ccgtgaaata gtacttcaca cccactaaag tgactattat
1081 aaaaacaaaa caaacaaaca aacaaaagat ggaaagtaac aagtgttggc caggatatag
1141 agaagttgga actcatgtag gttgctggtg ggaatgtaaa atggcacagc agctgtgtaa
1201 aaaagttttg tgattccttg cacagttaaa catagaatta ccgtatgatc tagcaattta
1261 acttctcagt atataccaaa aagaattgaa agcagggact caaacagata cttaacaatg
1321 tttatagcag cattattcaa aatagccaaa aggtggaaac aacacaaatg tccatcaaca
1381 gataaatgca taaacaaatt gtagcctgta catacgatgc aatattacac agcttaaaaa
1441 tgtaatgaaa ttctgacaca tgccacaaca tggatgaacc ttgaaaacat taggctaaga
1501 gaaataagct agacccaaaa gcacaaatat tgaatggttc cacttatatg aaataattag
1561 aataggcaaa ttcataggga aagacagtag agtgacagtt accaggtgct ggaggagagg
1621 gggaatggat agttttgggg tacagagttc tgtttggaat gaccaggagt tctgaaaatg
1681 gtcacacaac attatggtta atgccactga attgtgccct taagaatttt ttaaggagta
1741 aatttcatcg tgtgtgtatt ttgccacaat tttaaaaaca tcacttttcc ataaaaaaat
1801 aactagagac ccctgaacat acactgtgga cagattttat taataaatct tatggtttaa
1861 gtatcaagct attgttaatt acttctccta agtaaataaa cctaatacct ggtaaaatga
1921 tgtctagtta tgtggttgca gtatattaac caaggaggac tcgttgtttt gaagcaat
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
tgacttcagg atctggactt acc
<210> 3
<211> 22
<212> DNA
<213>artificial sequence
<400> 3
tgggaaatgt ggcaatgttg tt
Claims (10)
1. a long-chain non-coding RNA ENST00000509938.1 for high expressed in patients of acute myocardial infarction blood, its
It is characterised by that the sequence of this long-chain non-coding RNA ENST00000509938.1 is as shown in SEQ ID NO:1.
The long-chain non-coding of high expressed in patients of acute myocardial infarction blood the most according to claim 1
RNAENST00000509938.1, it is characterised in that this long-chain non-coding lncRNA ENST00000509938.1 is
The total length of ENST00000509938:1.
3. long-chain non-coding RNA ENST00000509938.1 according to claim 1 and 2 in a vitro detection blood
Preparation or diagnostic agent or medicine or test kit, it is characterised in that said preparation or diagnostic agent or medicine or test kit include specificity
Primer to, standard DNA template, PCR reactant liquor, wherein, specific primer is to including forward primer and downstream primer, forward primer
Nucleotides sequence be classified as SEQ ID No:2, the nucleotides sequence of downstream primer is classified as SEQ IDNo:3.
The preparation of long-chain non-coding RNA ENST00000509938.1 in vitro detection blood the most according to claim 3
Or diagnostic agent or medicine or test kit, it is characterised in that this test kit is fluorescent quantificationally PCR detecting kit.
5. according to the system of long-chain non-coding RNA ENST00000509938.1 in the vitro detection blood described in claim 3 or 4
Agent or diagnostic agent or medicine or test kit, it is characterised in that specific primer is to for SYBR Green, Taqman probe, molecule
Beacon, double cross probe, the detection of combined probe.
6. according to long-chain non-coding RNA ENST00000509938.1 in the vitro detection blood described in claim 3 or 4 or 5
Preparation or diagnostic agent or medicine or test kit, it is characterised in that PCR reactant liquor is fluorescence quantitative PCR reaction solution.
The preparation of long-chain non-coding RNA ENST00000509938.1 in vitro detection blood the most according to claim 6
Or diagnostic agent or medicine or test kit, it is characterised in that fluorescence quantitative PCR reaction solution includes dNTP, Mg2+, Taq enzyme.
8. according to long-chain non-coding in the vitro detection blood described in claim 3 or 4 or 5 or 6 or 7
The preparation of RNAENST00000509938.1 or diagnostic agent or medicine or test kit, it is characterised in that also include fluorescent dye.
9. the method detecting long-chain non-coding RNA ENST00000509938.1 according to claim 1 and 2, its
It is characterised by carrying out in the steps below: the first step, extracts blood total serum IgE；Second step, prepares cDNA；3rd step, quantitative amplification
lncRNA ENST00000509938.1。
10. a long-chain non-coding RNA ENST00000509938.1 according to claim 1 and 2 is used for examining in preparation
The test kit surveying acute myocardial infarction or the application of the novel targets being used as detection acute myocardial infarction medicine.
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