CN107447008A - For the enhancer RNA combinations of acatalepsia reason recurrent miscarriage, primer sets and application and kit - Google Patents
For the enhancer RNA combinations of acatalepsia reason recurrent miscarriage, primer sets and application and kit Download PDFInfo
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Abstract
The invention belongs to genetic engineering field, is related to the enhancer RNA combinations for acatalepsia reason recurrent miscarriage, primer sets and application and kit.The enhancer RNA combinations include：With SEQ ID NO：Nucleotide sequence shown in 1 has the first enhancer RNA and SEQ ID NO of more than 90% homology：Nucleotide sequence shown in 2 have more than 90% homology the second enhancer RNA and with SEQ ID NO：Nucleotide sequence shown in 3 has the 3rd enhancer RNA of more than 90% homology.The present invention using the enhancer RNA groups filtered out be used as new mark, prepare for unexplained recurrent spontaneous abortion diagnose or monitor kit, with high sensitivity, detect pedigree extensively, low cost and other advantages.
Description
Technical field
The invention belongs to genetic engineering field, more particularly, to the enhancing for acatalepsia reason recurrent miscarriage
The application and kit of sub- RNA combinations, primer sets, and enhancer RNA combinations with primer sets.
Background technology
Recurrent miscarriage (recurrent spontaneous abortion, RSA) refers to clinically continuous 2 times or 2 times
Before pregnant 20W above or pregnancy loss of the fetal weight less than 500g, its incidence account for the 1%~3% of pregnancy outcome.RSA
The cause of disease it is extremely complex, the clear and definite cause of disease has chromosome abnormality, reproductive anatomy exception, endocrinopathy, reproduction at present
System infections, autoimmunity and environmental factor etc., but the RSA for clinically still having 50%~60% can not find the clear and definite cause of disease, claim
For unexplained recurrent spontaneous abortion (unexplained recurrent spontaneous abortion, URSA).Embryo sends out
It is the main reason for URSA occurs to educate abnormal.Embryonic development is complex processes of the organism from the starting of its life to maturation, by one
The regulation and control of serial environmental factor, inherent cause and epigenetic modification.
In recent years, the discovery of long-chain non-coding RNA (long non-coding RNA, lncRNA) important biomolecule function
Become a large focal spot of life science, they can epigenetic, transcription and transcription after etc. many levels up-regulation
Control the expression of gene.In whole gene group transcription product, the ratio shared by the ratio considerably beyond coding RNA shared by lncRNA
Example.Different from coding RNA, lncRNA conservative is very different, but in its intramolecule, but containing more conservative part
Section, and its expression has Space-time speciality, these phenomenons have all prompted lncRNA to have important biological function.
After lncRNA is combined with transcription factor, lncRNA can form chromatin ring so as to regulate and control target base with target gene
The spatial and temporal expression of cause.Greenberg has found in mouse Nerve cell within 2010, is distributed in whole gene group, by neural thin
The enhancer that born of the same parents' unpolarizing is activated is transcribed, and lncRNA and the neighbouring gene of enhancer caused by enhancer transcription
The mRNA transcribed is associated.The researchs such as Rosenfeld find that in breast cancer cell, estrogen is combined with its acceptor and can lured
LncRNA transcription is led, and then improves the expression of specific gene, these lncRNA are removed by RNAi technology, enhancer can be weakened
The ability of up-regulated gene expression, while reduce the cyclic phenomenon between enhancer and promoter.In terms of embryonic development, lncRNA
Effect be also just gradually proved.Numerous studies show that lncRNA is critical turn in embryonic development and disease generating process
Record element, it may be possible to determine one of earliest period molecule of gene spatial and temporal expression in embryonic development, participate in regulation and control and grow correlation
Gene.Samir etc. reports that embryonic heart development is regulated and controled by lncRNA and its target gene, and lncRNA is centripetal in embryonic stem cell
Certain effect has been played in myocyte's atomization.
LncRNA is transcribed from " gene desert area " mostly, but some can be transcribed from the enhancer on genome
(enhancer) element, this kind of lncRNA are referred to as enhancer RNA (enhancer RNA, eRNA).Enhancer is can be with DNA
The region that transcription factor combines, can be located at upstream region of gene, may be alternatively located at downstream of gene, even remotely from modulated gene, and
It is often accompanied by the different modifying of histone.After being combined with transcription factor, enhancer can with target gene formed chromatin ring from
And regulate and control the spatial and temporal expression of target gene.
There is presently no the report that enhancer RNA is applied to unexplained recurrent spontaneous abortion auxiliary diagnosis, if can screen
Go out the related enhancer RNA of unexplained recurrent spontaneous abortion as biomarker, and develop corresponding diagnostic kit, to not
The diagnosis present situation of bright reason recurrent miscarriage will be once strong promotion, also be its drug screening, evaluating drug effect and targeting
Treatment opens up a new way.
The content of the invention
It is an object of the invention to provide for acatalepsia reason recurrent miscarriage enhancer RNA combination, primer sets and
Using and kit.
The present invention passes through difference point using the expression of enhancer RNA in high throughput sequencing technologies detection chorionic villi
Analysis screens lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 in unexplained recurrent spontaneous abortion chorionic villi sample
Expression significantly rise；Fluorescent quantitative PCR experiment further confirms lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 not
Expression quantity in bright reason patients with recurrent spontaneous abortion chorionic villi and serum is significantly higher than normal control.Pass through biological information credit
Analysis and ChIP-qPCR determine that lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are enhancer RNA.Pass through independent people
Group's checking and stability analysis, this group of enhancer RNA can be used as unexplained recurrent spontaneous abortion diagnosis marker in not clear original
Because being applied in the diagnosis of recurrent miscarriage.
Specifically, the first aspect of the present invention provides one group of enhancer RNA for being used for acatalepsia reason recurrent miscarriage
Combination, the enhancer RNA combinations include：With SEQ ID NO：Nucleotide sequence shown in 1 has the of more than 90% homology
One enhancer RNA and SEQ ID NO：Nucleotide sequence shown in 2 have more than 90% homology the second enhancer RNA and with
SEQ ID NO：Nucleotide sequence shown in 3 has the 3rd enhancer RNA of more than 90% homology.
Preferably, the enhancer RNA combinations include：With SEQ ID NO：Nucleotide sequence shown in 1 has more than 95%
First enhancer RNA of homology and SEQ ID NO：Nucleotide sequence shown in 2 has the second enhancing of more than 95% homology
Sub- RNA and with SEQ ID NO：Nucleotide sequence shown in 3 has the 3rd enhancer RNA of more than 95% homology.
It is further preferred that the enhancer RNA combinations include：With SEQ ID NO：Nucleotide sequence has shown in 1
96%th, the first enhancer RNA of 97%, 98%, 99% homology and SEQ ID NO：Nucleotide sequence shown in 2 have 96%,
97%th, the second enhancer RNA of 98%, 99% homology and with SEQ ID NO：Nucleotide sequence shown in 3 have 96%,
97%th, the 3rd enhancer RNA of 98%, 99% homology.
Most preferably, the enhancer RNA combinations include：With such as SEQ ID NO：First of nucleotide sequence shown in 1
Enhancer RNA, there is such as SEQ ID NO：Second enhancer RNA of nucleotide sequence shown in 2 and with such as SEQ ID NO：3 institutes
Show the 3rd enhancer RNA of nucleotide sequence.
The enhancer RNA combinations of the present invention can be as the biomolecule mark of acatalepsia reason recurrent miscarriage.
The second aspect of the present invention provides the primer sets for detecting above-mentioned enhancer RNA combinations, and the primer sets include：
For detecting the first enhancer RNA sense primer and anti-sense primer；
For detecting the second enhancer RNA sense primer and anti-sense primer；
For detecting the 3rd enhancer RNA sense primer and anti-sense primer.
Those skilled in the art can design above-mentioned primer according to conventional meanses, it is preferable that for detecting the first enhancer RNA
Sense primer there is SEQ ID NO：Nucleotide sequence shown in 4, anti-sense primer have SEQ ID NO：Nucleotides sequence shown in 5
Row；
Sense primer for detecting the second enhancer RNA has SEQ ID NO：Nucleotide sequence shown in 6, anti-sense primer
With SEQ ID NO：Nucleotide sequence shown in 7；
Sense primer for detecting the 3rd enhancer RNA has SEQ ID NO：Nucleotide sequence shown in 8, anti-sense primer
With SEQ ID NO：Nucleotide sequence shown in 9.
The present invention also provides above-mentioned enhancer RNA combinations and/or primer sets and examined preparing unexplained recurrent spontaneous abortion
Application in disconnected or monitoring reagent.
The third aspect of the present invention provides a kind of unexplained recurrent spontaneous abortion diagnosis or monitoring reagent box, the kit bag
Include above-mentioned primer sets.
The kit can also include internal reference, and other reagents that related round pcr is conventional.Preferably, the examination
Agent box also includes：
(1) GAPDH forward and reverse primer is detected；And/or
(2) archaeal dna polymerase, PCR buffer solutions, MgCl2, dNTPs, at least one of water and nucleic acid dye.
, can be by detecting above-mentioned enhancer RNA combinations in unexplained recurrent spontaneous abortion sample come real according to the present invention
Now diagnose or monitor, specific method includes but is not limited to hybrid method, TRAP and PCR sequencing PCR, and the hybrid method is that Northern is miscellaneous
Hand over, gene chip hybridization and hybridization in situ, the TRAP is Fluorescent quantitative PCR, the not clear original
Because recurrent miscarriage sample is unexplained recurrent spontaneous abortion decidua, fine hair, serum, blood plasma, oral cavity mucus, urine or body fluid.
Beneficial effects of the present invention include：
(1) present invention filters out one group of new enhancer RNA, this group of RNA and diagnosed available for unexplained recurrent spontaneous abortion
Specific marker.
(2) one group has been screened in unexplained recurrent spontaneous abortion and normal pregnant fine hair group by high throughput sequencing technologies
Knit the enhancer RNA of middle differential expression.
(3) using filter out one group of enhancer RNA as new mark, prepare and examined for unexplained recurrent spontaneous abortion
Disconnected or monitoring kit, has that high sensitivity, detection pedigree be wide, low cost and other advantages.
(4) marks of one group of enhancer RNA as unexplained recurrent spontaneous abortion is used, single mark will be improved
It is difficult to muting sensitivity caused by the individual difference that overcomes.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings.
Fig. 1 is that enhancer rna expression difference degree is sequenced in unexplained recurrent spontaneous abortion chorionic villi sample transcript profile
MA schemes.(it is more than 0 lncRNA for up-regulation in the lncRNA distributions that Control groups and Case groups inter-sample difference are expressed, is less than
0 lncRNA for downward)；
Fig. 2 is that Real-time PCR detections lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 answer in unknown cause
Expression schematic diagram in hair property miscarriage chorionic villi sample；
Fig. 3 is that Real-time PCR detections lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 answer in unknown cause
Expression schematic diagram in hair property miscarriage serum sample；
Fig. 4 is normal pregnancy control group and unexplained recurrent spontaneous abortion case group ROC curve.
Embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe being preferable to carry out for the present invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without should be limited by embodiments set forth herein.
Test method used in following embodiments is this area conventional method unless otherwise specified, or according to examination
Condition proposed by agent business is carried out with step.
Embodiment 1：Unexplained recurrent spontaneous abortion enhancer RNA is screened, screens and confirmed
1st, material
1.1 tissue
The clinical tissue sample of the present invention is all from March, 2015 in July, 2016 in the attached Huaian of Nanjing Medical University
The unexplained recurrent spontaneous abortion patient of First People's Hospital's diagnosis, control group stop pregnant for selection operation after same period normal pregnancy
The person of being pregnent simultaneously matches by strict.The use of all samples signs informed consent form by patient or its trustee, has obtained Nanjing
Medical university's Medical Ethics Committee standard.
1.2 reagent
TRIzolTMReagent is purchased from Invitrogen companies of the U.S..Reverse transcription reagent box (DRR036A) is purchased from Japan
Takara companies.SYBR reagents used in (Real-time) quantitative PCT only praise the limited public affairs of biotechnology to fluorescence purchased from Nanjing promise in real time
Department.PCR primer is designed and synthesized by Shanghai Ying Jun Bioisystech Co., Ltd.Transcript profile is sequenced and data analysis work is by Shanghai
Complete Tian Hao bio tech ltd.
2nd, method and result
2.1 unexplained recurrent spontaneous abortion chorionic villi sample transcript profiles are sequenced
Using the normal control sample that 3 unexplained recurrent spontaneous abortion patient chorionic villis and 3 processes strictly match as
Object, commission Shanghai Tianhao Biotech Co., Ltd. carry out transcript profile examining order：According to RNA extracts reagents TRIzolTM
The step of Reagent specifications are recommended extracts appearance total tissue RNA.After RNA sample quality inspection is qualified, using rRNA is removed, it is enriched with
Remaining mRNA, long noncoding RNA methods build sequencing library；Using Illumina Hiseq2500 high-flux sequences
Platform completes transcript folder RNA sequencing；100 bases are respectively surveyed at two-way paired end sequencing, i.e. 2 × 125bp, every reads both ends；
Average amount is not less than the every samples of 10G/ after Quality Control；Contamination control：RRNA ratios are less than the 3% of rawdata.
By being arranged to initial data, identify the potential enhancer RNA of differential expression, find lnc-ERGIC1-4,
Lnc-PBK-2, lnc-SLC4A1-1 significantly high expression (as shown in Figure 1) in unexplained recurrent spontaneous abortion chorionic villi.Its
Middle lnc-ERGIC1-4 transcript regions are located at No. 5 chromosomes, total length 344bp, nucleotide sequence such as SEQ ID NO：Shown in 1；
Lnc-PBK-2 transcript regions are located at No. 8 chromosomes, total length 267bp, nucleotide sequence such as SEQ ID NO：Shown in 2；lnc-
SLC4A1-1 transcript regions are located at No. 17 chromosomes, total length 460bp, nucleotide sequence such as SEQ ID NO：Shown in 3.
2.2 enhancer RNA confirm
Screening enhancer RNA is predicted by UCSC genome browser, finds lnc-ERGIC1-4, lnc-
PBK-2, lnc-SLC4A1-1 are transcribed from the enhancer region that there is H3K4me1 and H3K27ac to modify.Pass through ChIP-qPCR
When confirming that lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are overexpressed, H3K27ac expression raises, therefore confirms
Lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are enhancer RNA.
Embodiment 2：QRT-PCR method validation transcript profile sequencing results
1st, material
1.1 tissue
The clinical tissue sample of the present invention is the same as embodiment 1.
1.2 reagent
QPCR agents useful for same is the same as embodiment 1.
2nd, method
2.1 prepare cDNA samples
It will be organized in liquid nitrogen and grind, every 50~100mg tissues add 1mL TRIzol, are carried out with Syrup-homogenizing instrument at homogenate
Reason.TRIzol volumes 10% are not to be exceeded in sample volume.Homogenised sample is placed into 5min for (15~30 DEG C) in room temperature, makes nucleic acid egg
White compound is kept completely separate.0.2mL chloroforms often are added using 1mL TRIzol, acutely vibrate 15s, room temperature places 3min.4 DEG C,
10 000g centrifuge 15min.Sample is divided into 3 layers：Bottom is red organic phase, and upper strata is colourless aqueous phase and an intermediate layer.RNA
Mainly in aqueous phase, aqueous phase volume is about the 50~60% of TRIzol reagents used, and aqueous phase is transferred in new pipe.Use isopropanol
Precipitate the RNA in aqueous phase.0.5mL isopropanols often are added using 1mL TRIzol, fully vibration is mixed, and -20 DEG C of refrigerators are placed
20min.4 DEG C, 10000g centrifugation 10min, RNA precipitate is not seen before centrifugation, gelatinous precipitate occurs in pipe side and ttom of pipe after centrifugation.
Remove supernatant.RNA precipitate is washed with 75% fresh ethanol.Often at least add the ethanol of 1mL 75% using 1mL TRIzol.4 DEG C are not
5min is centrifuged more than 7500g, abandons supernatant.Room temperature places dry or vacuum and drains RNA precipitate, about dries in the air half an hour.Add
50 μ L DEPC water, inhaled and beaten several times with pipette tips, placing 15min for 37 DEG C dissolves RNA.Concentration, extraction are determined with Nanodrop2000
RNA be placed in -70 DEG C of preservations.
The RNA of extraction carries out reverse transcription reaction using 036A kits and obtains cDNA.The reaction system of reverse transcription includes
1000 μ g RNA sample, 4 μ 5 × Primescript of L Buffer, 1 μ L PrimerScript RT Enzyme MixI, 1 μ L
Oligo dT primer, 4 μ L Random 6mers, add DEPC water and system is complemented into 20 μ L.Reverse transcription reaction step is
37 DEG C are incubated 15 minutes, and 85 DEG C are incubated 5 seconds, you can obtain cDNA.The cDNA can be used for enhancer RNA Real-time PCR
Detection.
2.2 design of primers
Primer is designed and synthesized by Shanghai Ying Jun Bioisystech Co., Ltd：
Sequence is SEQ ID NO：The sense primer of 1 mark is SEQ ID NO：4(5’-
ATGTCCTCCACAATGTTAGCAA-3 '), anti-sense primer is SEQ ID NO：5(5’-TCTCGAACTCCTGACCTATCTTT-
3’)；
Sequence is SEQ ID NO：The sense primer of 2 mark is SEQ ID NO：6(5’-
TCATTCATTGATCTTCCCACAC-3 '), anti-sense primer is SEQ ID NO：7(5’-CCGTAGACAACTGGACACTCAG-
3’)；
Sequence is SEQ ID NO：The sense primer of 3 mark is SEQ ID NO：8(5’-
CCGTAGACAACTGGACACTCAG-3 '), anti-sense primer is SEQ ID NO：9(5’-TCATATTCCTCCTGCTCCAGAT-
3’)。
2.3qRT-PCR
Dye method：1 μ l cDNA are taken, by cDNA doubling dilutions, add 0.3 μ l Taq enzymes, 1 μ l 20 × EVA GREEN,
0.25 10 μM of μ l sense primers, 0.25 10 μM of μ l anti-sense primers, 1.2 μ l 25mM MgCl2, 1.6 μ l 2.5mM dNTP mixing
Liquid, 2 μ l 10 × PCR buffer solutions, 12.4 μ l pure water, 20 μ l systems carry out quantitative fluorescent PCR.Instrument uses ABI Prism
7900 quantitative real time PCR Instruments, PCR reaction condition are：95 DEG C of 5 minutes pre-degeneration → 95 DEG C are annealed for 15 seconds, and 60 DEG C extend for 1 minute
40 circulations are carried out, obtain ct values.Equation 2 can be used–△ctCalculate enhancer RNA relative expression quantities, wherein △ ct=cteRNA–
ctGAPDH。
3 results
To verify transcript profile sequencing result, unexplained recurrent spontaneous abortion chorionic villi sample of the present invention to clinical acquisitions
Carry out qRT-PCR detections (30:30), it was demonstrated that lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are in relapsed patient's suede
High expression (as shown in Figure 2), prompts lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 to participate in adjusting in hair tissue
The generating process of unexplained recurrent spontaneous abortion is controlled, there is the possibility applied to unexplained recurrent spontaneous abortion diagnosis and treatment.
Embodiment 3：Enhancer RNA lnc-ERGIC1-4, lnc-PBK-2, lnc- in qRT-PCR methods detection serum
SLC4A1-1 expressions
1st, material
1.1 serum
The clinical serum collection of specimens of the present invention is the same as embodiment 1.
1.2 reagent
QPCR agents useful for same is the same as embodiment 1.
2nd, method
1mL TRIzol are added in 200 μ l serum samples, are fully mixed, 5min is stood in 4 DEG C, makes nucleic acid-protein
Compound is kept completely separate.Subsequent processes are the same as embodiment 1 2.2.
3rd, result
Compared with normal pregnant, lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are in relapsed patient's serum
In high expression (as shown in Figure 3), prompt this group of enhancer RNA related to unexplained recurrent spontaneous abortion, available for the disease
The diagnosis of disease.
Embodiment 4：Enhancer RNA lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 combination are answered unknown cause
The judgement of hair property miscarriage
According to embodiment 2, the material of embodiment 3 and method, pass through the enhancer RNA to case and control group blood serum sample
The analysis of expression, using five quantiles of normal person's pregnancy controls enhancer rna expression amount as threshold value, to lnc-ERGIC1-
4th, lnc-PBK-2, lnc-SLC4A1-1 are scored, and further try to achieve total score, are drawn ROC curve with this and are carried out assessment prediction
Sensitivity and specificity, and then assess evaluation capacity of these three enhancer rna expressions to unexplained recurrent spontaneous abortion.ROC
Analysis result shows that lnc-ERGIC1-4, lnc-PBK-2, lnc-SLC4A1-1 are with 77.1% AUC (area under ROC curve)
Normal pregnancy control group and unexplained recurrent spontaneous abortion case group are separated into (as shown in Figure 4), the susceptibility of best cut point
=0.781, specificity=0.756.
On the basis of a series of above-mentioned results of study, it was demonstrated that use lnc-ERGIC1-4, lnc-PBK-2, lnc-
SLC4A1-1 well can separate normal pregnancy control group and unexplained recurrent spontaneous abortion case group.
Embodiment 5：Enhancer RNA diagnostic kits related to risk assessment are monitored for unexplained recurrent spontaneous abortion
Making
The manufacture craft and operating process of enhancer RNA kits are based primarily upon qRT-PCR technologies.
Unexplained recurrent spontaneous abortion patient and normal pregnancy collator's blood are determined by sequencing and qRT-PCR methods first
There is the enhancer RNA of a copy above in clear.Then the technology screenings such as quantitative PCR and unexplained recurrent spontaneous abortion phase are passed through
A kind of serum enhancer RNA of pass, the index as unexplained recurrent spontaneous abortion monitoring and risk assessment.Finally filter out pair
In 3-4 bars, this is simplifying for the optimization made on the basis of preliminary experiment for the serum enhancer RNA answered quantity control.This
Kit includes a collection of serum enhancer RNA primers, and wherein enhancer RNA primer includes lnc-ERGIC1-4, lnc-PBK-
2nd, lnc-SLC4A1-1 and GAPDH forward and reverse primer (being shown in Table 1).Also include related round pcr commonly use reagent, as Taq enzyme,
PCR buffer solutions, MgCl2, triphosphoric acid base deoxynucleotide mixed liquor, reagent, these reagents such as dyestuff can use corresponding city
Sell product.The value of this kit is only to need once to provide a small amount of (2ml) blood, you can detection serum enhancer RNA marks
The variation tendency of will thing, then the possibility occurred by the trend unexplained recurrent spontaneous abortion, and be easy to carry out
Dynamic monitoring.
Specific kit forms are as follows：
Kit contains following 3 pairs of primers：SEQ ID NO：4 and SEQ ID NO：5, SEQ ID NO：6 and SEQ ID
NO：7, SEQ ID NO：8 and SEQ ID NO：9, each 10 μM of 0.25 μ l.
0.3 μ l Taq enzymes, 1 μ 20 × EVA of l GREEN, 1.2 μ l 25mM MgCl can also be contained in kit2, 1.6 μ
L 2.5mM dNTP mixed liquors, 2 μ l 10 × PCR buffer solutions, 12.4 μ l pure water.
Forward and reverse primer a pair (tables 1) that can also be containing internal reference GAPDH in kit.Component in kit removes primer
Outer reagent can use the corresponding reagent for being used for the detection of enhancer rna content in the prior art.
1 embodiment of table, 5 related enhancer RNA primer sequence
It is described above various embodiments of the present invention, described above is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.In the case of without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes will be apparent from for the those of ordinary skill in art field.
Sequence table
<110>Nanjing Medical University
<120>For the enhancer RNA combinations of acatalepsia reason recurrent miscarriage, primer sets and application and kit
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<213> Artificial Sequence
<400> 10
cggagtcaac ggatttggtc gtat 24
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 11
agccttctcc atggtggtga agac 24
Claims (9)
1. one group of enhancer RNA for being used for acatalepsia reason recurrent miscarriage is combined, it is characterised in that the enhancer RNA
Combination includes：With SEQ ID NO：Nucleotide sequence shown in 1 has the first enhancer RNA and SEQ of more than 90% homology
ID NO：Nucleotide sequence shown in 2 have more than 90% homology the second enhancer RNA and with SEQ ID NO：Nucleosides shown in 3
Acid sequence has the 3rd enhancer RNA of more than 90% homology.
2. enhancer RNA combinations according to claim 1, wherein, the enhancer RNA combinations include：With SEQ ID
NO：Nucleotide sequence shown in 1 has the first enhancer RNA and SEQ ID NO of more than 95% homology：Nucleotides sequence shown in 2
Row with more than 95% homology the second enhancer RNA and with SEQ ID NO：Nucleotide sequence shown in 3 has more than 95%
3rd enhancer RNA of homology.
3. enhancer RNA combinations according to claim 1, wherein, the enhancer RNA combinations include：With such as SEQ
ID NO：First enhancer RNA of nucleotide sequence shown in 1, there is such as SEQ ID NO：Second of nucleotide sequence shown in 2 increases
Hadron RNA and with such as SEQ ID NO：3rd enhancer RNA of nucleotide sequence shown in 3.
4. the enhancer RNA in claim 1-3 described in any one combines the life as acatalepsia reason recurrent miscarriage
Thing molecular marker.
5. the primer sets of the enhancer RNA combinations in 1-3 described in any one are required for test right, it is characterised in that institute
Stating primer sets includes：
For detecting the first enhancer RNA sense primer and anti-sense primer；
For detecting the second enhancer RNA sense primer and anti-sense primer；
For detecting the 3rd enhancer RNA sense primer and anti-sense primer.
6. primer sets according to claim 5, wherein,
Sense primer for detecting the first enhancer RNA has SEQ ID NO：Nucleotide sequence shown in 4, anti-sense primer have
SEQ ID NO：Nucleotide sequence shown in 5；
Sense primer for detecting the second enhancer RNA has SEQ ID NO：Nucleotide sequence shown in 6, anti-sense primer have
SEQ ID NO：Nucleotide sequence shown in 7；
Sense primer for detecting the 3rd enhancer RNA has SEQ ID NO：Nucleotide sequence shown in 8, anti-sense primer have
SEQ ID NO：Nucleotide sequence shown in 9.
7. the enhancer RNA combinations described in any one and/or the primer sets described in claim 5 or 6 exist in claim 1-3
Prepare the application in unexplained recurrent spontaneous abortion diagnosis or monitoring reagent.
8. a kind of unexplained recurrent spontaneous abortion diagnosis or monitoring reagent box, it is characterised in that the kit includes claim 5
Or the primer sets described in 6.
9. kit according to claim 8, wherein, the kit also includes：
(1) GAPDH forward and reverse primer is detected；And/or
(2) archaeal dna polymerase, PCR buffer solutions, MgCl2, dNTPs, at least one of water and nucleic acid dye.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486241A (en) * | 2018-03-13 | 2018-09-04 | 南京医科大学 | Serum messenger RNA biomarker, primer sets and application for diagnosing recurrent miscarriage and kit |
| CN113436741A (en) * | 2021-07-16 | 2021-09-24 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
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| WO2013109145A1 (en) * | 2012-01-18 | 2013-07-25 | Stichting Katholieke Universiteit | Annexin a5 snp |
| WO2014160032A1 (en) * | 2013-03-13 | 2014-10-02 | University Of Louisville Research Foundation, Inc. | Exosome-associated rna as a diagnostic marker |
| CN106319031A (en) * | 2015-06-23 | 2017-01-11 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion relevant microRNA and applications thereof |
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| KR20120102216A (en) * | 2011-03-08 | 2012-09-18 | 차의과학대학교 산학협력단 | Analytical method for diagnosing recurrent pregnancy loss and kit therefor |
| WO2013109145A1 (en) * | 2012-01-18 | 2013-07-25 | Stichting Katholieke Universiteit | Annexin a5 snp |
| WO2014160032A1 (en) * | 2013-03-13 | 2014-10-02 | University Of Louisville Research Foundation, Inc. | Exosome-associated rna as a diagnostic marker |
| CN106319031A (en) * | 2015-06-23 | 2017-01-11 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion relevant microRNA and applications thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486241A (en) * | 2018-03-13 | 2018-09-04 | 南京医科大学 | Serum messenger RNA biomarker, primer sets and application for diagnosing recurrent miscarriage and kit |
| CN108486241B (en) * | 2018-03-13 | 2022-04-12 | 南京医科大学 | Serum messenger ribonucleic acid biomarker for diagnosing recurrent abortion, primer group, application and kit |
| CN113436741A (en) * | 2021-07-16 | 2021-09-24 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
| CN113436741B (en) * | 2021-07-16 | 2023-02-28 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
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