CN105112549B - A kind of microRNA molecule label miR-23a of quick detection sow Oocyte quality and its application - Google Patents
A kind of microRNA molecule label miR-23a of quick detection sow Oocyte quality and its application Download PDFInfo
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Abstract
A kind of application the invention discloses microRNA miR 23a as quick detection sow Oocyte quality, miRNA molecule marker overexpression in the low cumulus oocyte of potentiality of development, and it is secreted into serum by the active secretion process of tissue, then by detecting the low sow serum miR 23a expressions of large sample Growth Results, it is found that serum miR 23a are a kind of molecular marked compounds that can quickly detect sow Oocyte quality；The present invention predicts sow reproductive potential by detecting Oocyte quality, eliminates the low sow of production potential as early as possible, this will substantially reduce feed and human cost, contribute to flourishing for China's live pig industry.
Description
Technical field
The invention belongs to technical field of molecular biology, are related to a kind of miRNA of quick detection sow Oocyte quality
Primer, kit and the detection method of molecular labeling miR-23a, amiR-23a.
Background technology
The economic benefit height on pig farm has basic relationship with sow breeding potential.The reproductive performance of sow is to weigh pig raising
A principal economic indicators in production.In pig production in addition to give full play to and using sow reproductive performance, it is also necessary to
The low sow of superseded production performance in time, could realize the maximization of economic benefit.How the bad mother of Growth Results is eliminated as early as possible
Pig becomes a difficult problem of puzzlement pig production.Research finds that sow Oocyte quality is positively correlated pass with Growth Results
System.Therefore by detecting the method that sow Oocyte quality is most accurate, most effective evaluation sow Growth Results.But at present
Laboratory stage is all rested on to sow Oocyte quality detection method, and sow can all be damaged, for production
Have no directive significance.
MiRNAs be a kind of length be 19-25 nucleotide non-coding tiny RNA, be highly conserved during evolution and
It is specific expressed in each histocyte.More and more researchs find that miRNAs participates in a variety of lifes by post-transcriptional control gene expression
The adjusting of object signal path.More and more research evidences have shown that miRNA is adjusting ovum mother carefully as a kind of regulatory factor
Play a significant role during born of the same parents' maturation, corpus luteum development, early embryonic development and embryo nidation etc., and much with ovary phase
Play the part of role of crucial importance during the occurrence and development of related disorders, can be used for predicting infertile, oophoroma, polycystic ovary synthesis
It levys raw molecular marker.
Research has confirmed that there are hundreds of kinds of miRNAs, these microRNAs s to be stable in the presence of in serum/plasma
In cycle, and it is not easy to be degraded by endogenous RNA enzyme.Studies have found that the serum and cell RNA extract that are handled through RNA enzyme,
The RNA of macromolecular all degrades, but miRNAs still preferably keeps stablizing and integrality, miRNAs can also resist a variety of severe outer
Environment such as boils, too high or too low pH value, multigelation, requires condition of storage also not stringent.MiRNAs is in same sample
Expression is kept constant, and is repeatedly detected same sample miRNAs contents, is as a result stablized.The technology of existing maturation, including it is fixed
Property and quantitative miRNA molecule technology, it is a kind of very accurate and effectively to show that serum miRNAs can be used as, and can be applicable in
The Molecular biomarkers of pig production.
There is result of study to show that miRNA molecule is obtained by the miRNA active secretions in organizing in serum, therefore
It can be changed by the express spectra of miRNA in the egg mother cell of high-throughput chip analysis potentiality of development difference to screen in serum
MiRNA, in conjunction with sow Growth Results, the miRNA levels variation of large sample analysis swinery serum abnormal expression.This will be a kind of
The method of very effective screening molecular marked compound.
Sow reproductive potential is predicted by detecting Oocyte quality, eliminates the low sow of production potential as early as possible, this will be big
It is big to reduce feed and human cost, contribute to flourishing for China's live pig industry.Therefore develop a kind of suitable pig farm, it is noninvasive simultaneously
The molecular marker that sow Oocyte quality can accurately be detected is most important.
Invention content
The present invention makes improvements in view of the above problems, proposes a kind of blood of quick detection sow Oocyte quality
Clear miRNA labels.
The present invention is achieved by the following technical solutions：
The present invention filters out overexpression by microRNA genetic chips from the low egg mother cell of potentiality of development
MicroRNA, and verified by realtime PCR, final clear miR-23a and Oocyte quality are highly relevant, Ke Yizuo
Quickly to detect the marker of Oocyte quality.
Because the miR-23a of overexpression can pass through the active secretion process of tissue in the low egg mother cell of potentiality of development
It is secreted into serum, (its sequence is so the selective analysis of the present invention ripe body of the low sow serum miR-23a of Growth Results
5 '-AUCACAUUGCCAGGGAUUUCC-3 ', such as SEQ ID NO：Shown in 1) expression, find serum miR-23a for the first time
Quick detection sow Oocyte quality molecular marker can be used as to be applied.When concrete application, occur in sow quiet vertical
The next morning of reflection acquires blood sample on an empty stomach, using the expression of miR-23a in Realtime PCR detection serum, if energy
Detection, then the sow should be eliminated.
To achieve the above object, miRNA points that the present invention provides a kind of for quickly detecting sow Oocyte quality
Son label miR-23a, the nucleotide sequence such as sequence table SEQ ID NO of the molecular labeling:Shown in 1, the molecular labeling
MiR-23a overexpressions in potentiality of development difference cumulus oocyte, and blood is secreted by the active secretion process of tissue
In clear；It secretes to the nucleic acid molecule label miR-23a in serum and is used for the quick detection of sow Oocyte quality.
To achieve the above object, the present invention also provides for detecting the molecular labeling miR-23a primer pairs, nucleotide
Sequence is as follows：
Forward primer：5 '-ATCACATTGCCAGGGATTTCC-3 ', as shown in SEQ ID NO.2；
Reverse primer sequence is general aptamer primer.
The primer pair specific amplification goes out SEQ ID NO:Nucleotide sequence shown in 1.
To achieve the above object, the present invention also provides a kind of reagents for quickly detecting sow Oocyte quality
Box, including above-mentioned primer pair.
The kit further includes 2.5U/ μ l Poly A polymerases, reverse transcriptase, 5 × RT Buffer, goes RNA enzyme
Water, 2 × qPCR mixed liquors, 70% ethyl alcohol, serum lysate, chloroform and absolute ethyl alcohol.
The concrete composition of the kit is as follows：
20 μ l of 2.5U/ μ l Poly A polymerases；
20 μ l of reverse transcriptase；
5 × RT Buffer, 100 μ l；
Remove 1000 μ l of RNA enzyme water；
2 × qPCR mixed liquors, 2000 μ l；
20 μ l of 50um upstream specific primers；
0 μ l of 50um lower adaptations primer 2；
20 μ l of internal reference upstream primer；
20 μ l of internal reference downstream primer；
Serum lysate 100ml；
70% ethyl alcohol 100ml；
Chloroform 100ml；
Absolute ethyl alcohol 100ml.
Use mentioned reagent box detection miR-23a specific method for：
(1) extraction of microRNA：
1. taking the 400 μ l of peripheral blood serum of sow, 750 μ l lysate MRL are added, piping and druming several times, acutely shakes mixing,
It is incubated 5min at room temperature, careful supernatant of drawing is transferred in the centrifuge tube of a new RNase free.
2. plus 1mL Trizol reagents, mixing are placed 10 minutes at room temperature.4 DEG C of 12000rpm are centrifuged 15 minutes.
3. supernatant is taken to be gone in the EP pipes of RNase to 1.5mL, 200 μ l chloroforms are added, shakes vigorously and mix well 30 seconds, makes
Water phase and organic phase can be sufficiently mixed, and be stored at room temperature 15 minutes, 4 DEG C of 12000rpm refrigerated centrifuges 10 minutes.
4. draw upper strata aqueous phase part move to another it is new go the EP of RNase manage, 0.5mL isopropanols are added, concussion mixes
10 minutes are stood after even at room temperature, 4 DEG C of 12000rpm refrigerated centrifuges 10 minutes.
5. removing supernatant, 70% ethyl alcohol is added to precipitate RNA compositions, and suitable DEPC water is added and is dissolved.
(2) detection of miR-23a：
1. going RNA enzyme according to obtained 5 μ l+ reverse transcriptases of RNA+ RT Buffers, 1 μ l+2.5U/ μ l polymerases, 1 μ l+
Water is supplied to 25 μ l.
2. 37 DEG C of water-bath 60min；It is put in the microRNAcDNA on ice, obtained after 85 DEG C of water-bath 5min.
10 μ l+ primers (2 μM) of cDNA2 μ l+2xqPCR mixed liquors 2 3. μ l+ universal primers (2 μM) 2 μ l+DDW, 4 μ l.
4. 95 DEG C of 10min of the pre-degeneration → cycle of (denaturation 15 seconds → annealing 60 DEG C 20 seconds → extends 72 DEG C 30 seconds) 40, knot
Melt curve analysis analysis is carried out after beam immediately, solubility curve selects auto-programming：Detailed process is：95 DEG C 15 seconds, 60 DEG C 1 minute,
95 DEG C 15 seconds, 60 DEG C 15 seconds.
5. according to obtained Ct values, reference gene is subtracted, 2- Δ Δ CT is substituted into, obtains miR-23a in the sow serum
Expression value.
To achieve the above object, the specificity that the present invention also provides a kind of for detecting serum miR-23a maturation bodies expands
The method of increasing, includes the following steps：The extracting of serum microRNA is carried out first, then in poly (A) polymerases at the ends its 3'
End plus polyA tails, reverse transcription is cDNA under the action of reverse transcriptase, and carries out Real timePCR amplifications.
To achieve the above object, the present invention also provides following any applications：
(1) application of the above-mentioned molecular labeling in detection Oocyte quality prediction sow reproductive potential；
(2) application of the above-mentioned primer pair in detection Oocyte quality prediction sow reproductive potential；
(3) application of the above-mentioned kit in detection Oocyte quality prediction sow reproductive potential；
(4) application of the above-mentioned method in detection Oocyte quality prediction sow reproductive potential.
Beneficial effects of the present invention：
The present invention is female using the high sow ovum of microRNA genechip detections and comparative development potential difference and potentiality of development
Cell microRNA express spectra differences, therefrom filter out candidate microRNA, are verified by realtime PCR, find
MiR-23a can be as the molecular marked compound of detection Oocyte quality.The miR-23a of overexpression can pass through tissue
Active secretion process is secreted into serum, therefore the then low sow serum miR-23a expression of large sample analysis Growth Results
Level finds that serum miR-23a is a kind of molecular marked compound that can quickly detect sow Oocyte quality.The present invention provides
Molecular marker of this miRNA molecule as quick detection sow Oocyte quality, to prediction sow production potential tool
It is significant.
Description of the drawings
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is the low cumulus oocyte of potentiality of development and Normal group miRNA expression analysis；
Fig. 2 is Oocyte quality difference sow and Normal group serum miRNA expression analysis.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts
Embodiment shall fall within the protection scope of the present invention.
The low cumulus oocyte miRNA express spectra variations of 1 chip method comparative development potential of embodiment
Market pigs ovary is acquired from slaughterhouse, ovary transports laboratory back as early as possible in 37 DEG C of sterile salines.With 20
The asepsis injector of number syringe needle extracts Ovarian surface diameter in the ovarian follicle of 2-6mm, and the liquor folliculi of extraction is slowly injected high pressure and is gone out
The centrifuge tube that bacterium crosses makes egg mother cell natural subsidence 10 minutes, and ovarian follicle supernatant is then slowly drawn with pipettor, will be taken off
Precipitation after clear is transferred in the 60mm culture dishes containing PBS buffer solution.It is with mouth suction pipe that ovarian cumulus ovum is female under stereomicroscope
Cell is divided into two classes and is transferred in 1ml centrifuge tubes, and -70 DEG C of preservations are to be measured.Criteria for classification：The low ovarian cumulus ovum of potentiality of development is female thin
Born of the same parents：Oocyte cytoplasm is uniform, only single layer cumulus cell or without cumulus cell be coated with.Normal group：Egg mother cell matter is equal
It is even, around there are 4 layers or more cumulus cell coatings.
RNA in cumulus oocyte is extracted by the following method.
1. cumulus oocyte is directly added into Trizol, 1mL Trizol solution is added by 0.1g, overturns mixing, room temperature is quiet
Set 5min；
2. adding 0.2mL chloroforms per 1mL Trizol.Sample tube cover is covered tightly, 15sec is firmly rocked, it is made to mix well, room
12000rmp centrifuges 15min after temperature stands 5min；
3. upper strata aqueous phase is transferred in new 1.5mLEP pipes, isometric isopropanol is added, is put in and is stored at room temperature after mixing
10min, 12000rmp centrifuge 10min；
4. abandoning supernatant, precipitation is placed on ventilation, is dried；
5. 10 65 DEG C of dissolutions of μ lDEPC water are added.
The expression chip analysis that the RNA of extraction is carried out to miRNA, the results are shown in Table 1.By being found in table 1, miR-23a is being sent out
Educating in the low cumulus oocyte of potential and Normal group expression has significant difference, the sequence such as sequence of the miR-23a
Shown in row 1, sequence is：5’-AUCACAUUGCCAGGGAUUUCC-3’(SEQ ID NO：1).The miR-23a specificity
Forward direction primer sequence is：5’-ATCACATTGCCAGGGATTTCC-3’(SEQ ID NO：2)；Reverse primer sequence is general
Aptamer primer.
The variation of 1 chip analysis potentiality of development of table low cumulus oocyte and Normal group miRNA express spectras
The result of the real-time round pcr proofing chips of embodiment 2
Market pigs ovary is acquired from slaughterhouse, ovary transports laboratory back as early as possible in 37 DEG C of sterile salines.With 20
The asepsis injector of number syringe needle extracts Ovarian surface diameter in the ovarian follicle of 2-6mm, and the liquor folliculi of extraction is slowly injected high pressure and is gone out
The centrifuge tube that bacterium crosses makes egg mother cell natural subsidence 10 minutes, and ovarian follicle supernatant is then slowly drawn with pipettor, will be taken off
Precipitation after clear is transferred in the 60mm culture dishes containing PBS buffer solution.It is with mouth suction pipe that ovarian cumulus ovum is female under stereomicroscope
Cell is divided into two classes and is transferred in 1ml centrifuge tubes, and -70 DEG C of preservations are to be measured.Criteria for classification：The low ovarian cumulus ovum of potentiality of development is female thin
Born of the same parents：Oocyte cytoplasm is uniform, only single layer cumulus cell or without cumulus cell be coated with.Normal group：Egg mother cell matter is equal
It is even, around there are 4 layers or more cumulus cell coatings.
RNA in cumulus oocyte is extracted by the following method：
1. cumulus oocyte is directly added into Trizol, 1mL Trizol solution is added by 0.1g, overturns mixing, room temperature is quiet
Set 5min；
2. adding 0.2mL chloroforms per 1mL Trizol.Sample tube cover is covered tightly, 15sec is firmly rocked, it is made to mix well, room
12000rmp centrifuges 15min after temperature stands 5min；
3. upper strata aqueous phase is transferred in new 1.5mLEP pipes, isometric isopropanol is added, is put in and is stored at room temperature after mixing
10min, 12000rmp centrifuge 10min；
4. abandoning supernatant, precipitation is placed on ventilation, is dried；
5. 10 65 DEG C of dissolutions of μ lDEPC water are added.
MicroRNA reverse transcriptions
2 μ g RNA are taken, are immediately placed on ice after thaw at RT, defrosting.Mixed liquor is prepared according to table 3.
The reaction system of 3 microRNA reverse transcriptions of table
37 DEG C, 60min；85 DEG C, it is put in the microRNA cDNA obtained on ice after 5min and can be used for subsequent experimental, or sets
In -20 DEG C of preservations.
Real-time PCR detect gene expression
The cDNA obtained using above-mentioned reverse transcription reaction is template, each each three multiple holes of template-setup of testing gene,
It operates on ice, reaction system is prepared as shown in table 4.
4 Realtime PCR reaction systems of table
Method PCR sets reaction step in two steps.Reaction terminates, and according to melting curve and Ct values, mesh is obtained according to formula
Relative Expression values of the gene in this template after analyze.The results are shown in Table 2,
The expression of miR-23a in the low cumulus oocyte of 2 real-time PCR detection potentiality of development of table and normal control
It is horizontal
| Sample number into spectrum | Cumulus oocyte type | MiR-23a relative expression levels |
| 1 | 0 | 1.1783 |
| 2 | 0 | 2.3591 |
| 3 | 0 | 1.6782 |
| 4 | 0 | 2.0390 |
| 5 | 0 | 1.4100 |
| 6 | 0 | 2.0180 |
| 7 | 0 | 2.7500 |
| 8 | 0 | 4.2901 |
| 9 | 0 | 3.3010 |
| 10 | 0 | 2.4700 |
| 11 | 1 | 50.6901 |
| 12 | 1 | 49.8961 |
| 13 | 1 | 51.5020 |
| 14 | 1 | 49.9000 |
| 15 | 1 | 42.0000 |
| 16 | 1 | 45.2040 |
| 17 | 1 | 55.1500 |
| 18 | 1 | 57.9012 |
| 19 | 1 | 53.3600 |
| 20 | 1 | 65.0020 |
In table 2, " 0 " indicates that normal control, " 1 " indicate the low cumulus oocyte of potentiality of development.
For statistical analysis through SPASS softwares, miR-23a expressions in the low cumulus oocyte of potentiality of development are notable
Higher than Normal group (P ＜ 0.05), the results are shown in Figure 1.
It is possible thereby to determine, there are significant differences in the low cumulus oocyte of potentiality of development by miR-23a, can be used as inspection
Survey the molecular labeling of Oocyte quality.
Embodiment 3 detects the expression of miR-23a in Oocyte quality difference sow serum with real-time round pcrs
It is horizontal
Serum sample is collected：Quiet vertical reflection the next morning occurs in sow, and acquisition 10ml blood is coagulated in rush in pipe on an empty stomach,
2000~3000rpm centrifuges 18~20min, draws supernatant in no RNase centrifuge tubes, 400 μ l packing, and -80 DEG C of freezings are to be measured.
Study sample selection criteria：Continuous two parity litter sizes are less than 5, and Body Condition Score (2.5-3.5), feeding is normally
The female Landrace of health is defined as Oocyte quality difference group；It is identical as Oocyte quality difference group parity, continuous two parity
Litter size 10-13 heads, Body Condition Score (2.5-3.5), normal healthy female Landrace of searching for food are defined as Normal group.It collects altogether
100 samples, wherein 50 come from low group of litter size, 50 come from Normal group.
The extraction of serum microRNA
Steps are as follows：
1. taking the 400 μ l of peripheral blood serum of sow, 750 μ l lysate MRL are added, piping and druming several times, acutely shakes mixing,
It is incubated 5min at room temperature, careful supernatant of drawing is transferred in the centrifuge tube of a new RNase free.
2. plus 1mL Trizol reagents, mixing are placed 10 minutes at room temperature.4 DEG C of 12000rpm are centrifuged 15 minutes.
3. supernatant is taken to be gone in the EP pipes of RNase to 1.5mL, 200 μ l chloroforms are added, shakes vigorously and mix well 30 seconds, makes
Water phase and organic phase can be sufficiently mixed, and be stored at room temperature 15 minutes, 4 DEG C of 12000rpm refrigerated centrifuges 10 minutes.
4. draw upper strata aqueous phase part move to another it is new go the EP of RNase manage, 0.5mL isopropanols are added, concussion mixes
10 minutes are stood after even at room temperature, 4 DEG C of 12000rpm refrigerated centrifuges 10 minutes.
5. removing supernatant, 70% ethyl alcohol is added to precipitate RNA compositions, and suitable DEPC water is added and is dissolved.
MicroRNA reverse transcriptions
2 μ g RNA are taken, are immediately placed on ice after thaw at RT, defrosting.Mixed liquor is prepared according to table 3.
The reaction system of 3 microRNA reverse transcriptions of table
37 DEG C, 60min；85 DEG C, it is put in the microRNA cDNA obtained on ice after 5min and can be used for subsequent experimental, or sets
In -20 DEG C of preservations.
Real-time PCR detect gene expression
The cDNA obtained using above-mentioned reverse transcription reaction is template, each each three multiple holes of template-setup of testing gene,
It operates on ice, reaction system is prepared as shown in table 4.
4 Realtime PCR reaction systems of table
Method PCR sets reaction step in two steps.Reaction terminates, and according to melting curve and Ct values, mesh is obtained according to formula
Relative Expression values of the gene in this template after analyze.The results are shown in Table 5,
5 real-time PCR of table detect the expression of miR-23a in the normal and low sow serum of litter size
In table 5, " 0 " indicates that Normal group (continuous two parity litter sizes 10-13), " 1 " indicate egg mother cell matter
The poor sow of amount (continuous two parity litter sizes are less than 5).
It is for statistical analysis through spass softwares, expression of the miR-23a in Oocyte quality difference group and Normal group
Level has significant difference (p<0.05), the results are shown in Figure 2.
It is possible thereby to determine, there are significant differences in Oocyte quality difference group sow serum by miR-23a, can conduct
Detect the molecular labeling of sow Oocyte quality.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Claims (3)
1. a kind of primer pair for detecting sow serum miR-23a expressions is female carefully for quickly detecting sow ovum in making
Application in the kit of cytoplasm amount, it is characterised in that：The nucleotide sequence of the primer pair is as follows：
Forward primer：5 '-ATCACATTGCCAGGGATTTCC-3 ', as shown in SEQ ID NO.2；Reverse primer sequence is general
Aptamer primer.
2. application according to claim 1, it is characterised in that：Further include 2.5U/ μ l Poly A polymerizations in the kit
Enzyme, 5 × RT Buffer, removes RNA enzyme water, 2 × qPCR mixed liquors, 70% ethyl alcohol, serum lysate, chloroform at reverse transcriptase
And absolute ethyl alcohol.
3. application according to claim 1, it is characterised in that：The concrete composition of the kit is as follows：
20 μ l of 2.5U/ μ l Poly A polymerases；
20 μ l of reverse transcriptase；
5 × RT Buffer, 100 μ l；
Remove 1000 μ l of RNA enzyme water；
2 × qPCR mixed liquors, 2000 μ l；
50 μM of 20 μ l of upstream specific primer；
50 μM of 0 μ l of lower adaptation primer 2；
20 μ l of internal reference upstream primer；
20 μ l of internal reference downstream primer；
Serum lysate 100ml；
70% ethyl alcohol 100ml；
Chloroform 100ml；
Absolute ethyl alcohol 100ml.
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| CN106978472A (en) * | 2016-01-15 | 2017-07-25 | 陈薪 | The related microRNA of human oocyte Quality Down and its application |
| CN106434912B (en) * | 2016-09-22 | 2021-05-18 | 安徽农业大学 | Application of microRNA marker related to litter size of pig |
| CN109082463B (en) * | 2018-09-03 | 2021-05-14 | 天津渤海水产研究所 | Cynoglossus semilaevis exosome microRNA for indicating sex and kit |
| CN110295222A (en) * | 2019-01-11 | 2019-10-01 | 安徽省农业科学院畜牧兽医研究所 | A kind of miRNA screening technique influencing pig number born character |
| CN110592232B (en) * | 2019-09-23 | 2022-04-26 | 四川农业大学 | Molecular marker related to pork pH value character and application thereof |
| CN111560443B (en) * | 2020-06-19 | 2022-11-08 | 湖南人文科技学院 | Group of biomolecular markers for screening high-quality porcine oocytes and application thereof |
| CN114875028B (en) * | 2022-05-27 | 2023-06-09 | 南京农业大学 | Mutation site associated with sow total litter size trait and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2693595A1 (en) * | 2007-07-13 | 2009-01-22 | Plumchoice, Inc. | Systems and methods for distributing remote technical support via a centralized service |
-
2015
- 2015-09-25 CN CN201510623895.9A patent/CN105112549B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| Cloning, characterization and expression analysis of porcine microRNA;Alavala Matta Reddy等;《BMC Genomics》;20090205;第10卷(第65期);第3页表1 * |
| Differentially expressed plasma microRNAs in premature ovarian failure patients and the potential regulatory function of mir-23a in granulosa cell apoptosis;Xiaokui Yang等;《Reproduction》;20120531;第144卷;第237页左栏第1段 * |
| Identification and characterization of new microRNAs from pig;Jung Kim等;《Mamm Genome》;20080612;第19卷;第570–580页 * |
| MicroRNAs in ovarian function and disorders;Ying Li等;《Journal of Ovarian Research》;20150801;第8卷(第51期);第1-8页 * |
| Regulatory role of microRNAs in ovarian function;Tal Imbar等;《Fertility and Sterility》;20140630;第101卷(第6期);第1524-1530页 * |
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