CN105112549A - MicroRNA (micro ribonucleic acid) molecular marker miR-23a for quickly detecting quality of oocytes of sows and application of microRNA molecular marker miR-23a - Google Patents
MicroRNA (micro ribonucleic acid) molecular marker miR-23a for quickly detecting quality of oocytes of sows and application of microRNA molecular marker miR-23a Download PDFInfo
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Abstract
The invention discloses application of a microRNA (micro ribonucleic acid)-miR-23a molecular marker miR-23a to quickly detecting the quality of oocytes of sows. The miRNA molecular marker miR-23a in the cumulus oocytes with low potentiality development is abnormally high in expression and can be secreted in serum via active secretion procedures of tissues, and serum miR-23a is the molecular marker with a function of quickly detecting the quality of the oocytes of the sows as discovered by large-sample detection on the expression level of the serum miR-23a of the sows with low production performance. The application has the advantages that the reproductive potential of the sows can be predicted by means of detecting the quality of the oocytes, so that the sows with low production potential can be eliminated as early as possible, the feed and labor costs can be greatly reduced, and the application is beneficial to vigorous development of the pig industry in China.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to the primer of miRNA molecule mark miR-23a, amiR-23a of a kind of rapid detection sow Oocyte quality, test kit and detection method.
Background technology
The economic benefit height on pig farm has basic relation with sow breeding potential.The reproductive performance of sow weighs a principal economic indicators in Swine Production.Except giving full play to and utilize the reproductive performance of sow in Swine Production, also need to eliminate the low sow of production performance in time, the maximization of economic benefit could be realized.How to eliminate the not good sow of Growth Results as early as possible, become a difficult problem of puzzlement Swine Production.Research finds that sow Oocyte quality becomes positive correlation with Growth Results.Therefore by detect sow Oocyte quality be the most accurately, the method for most effective evaluation sow Growth Results.But at present all laboratory stage is rested on to sow Oocyte quality detection method, and all can damage sow, directive significance be there is no for production.
MiRNAs is a class length is 19-25 Nucleotide non-coding tiny RNA, during evolution high conservative specific expressed in each histocyte.More and more research finds that miRNAs participates in the adjustment of various biological signal path by post-transcriptional control genetic expression.More and more research evidence has shown that miRNA plays a significant role in the processes such as adjustment oocyte maturation, corpus luteum development, early embryonic development and embryo nidation as a kind of regulatory factor, and in a lot of generation with ovary relative disease and evolution, play the part of role of crucial importance, can be used for predicting molecular marker that is infertile, the generation of ovarian cancer, polycystic ovarian syndrome.
Research confirmed the miRNAs that there is hundreds of kind in serum/plasma, these microRNAs s Absorbable organic halogens is present in circulation, and not easily degrade by endogenous RNA enzyme.Studies have found that the serum through RNA ferment treatment and cell RNA extract, macromolecular RNA all degrades, but miRNAs still keeps stable and integrity preferably, miRNAs also can resist multiple severe external environment as boiled, too high or too low pH value, multigelation, require also not strict to condition of storage.MiRNAs keeps constant at same sample expression level, repeatedly detects same sample miRNAs content, and result is stablized.Existing proven technique, comprises the technology of quantitative and qualitative analysis miRNA molecule, shows that serum miRNAs one very accurately with effective, and can be suitable for the Molecular biomarkers of Swine Production.
Result of study is had to show, in serum, miRNA molecule is obtained by the miRNA active secretion in tissue, therefore can change by the express spectra of miRNA in the ovocyte of high-throughput chip analysis potentiality of development difference the miRNA screened in serum, again in conjunction with sow Growth Results, the miRNA level change that large sample analysis swinery serum abnormal is expressed.This kind of method that will be very effective screening molecular marked compound.
By detecting Oocyte quality prediction sow reproductive potential, eliminate as early as possible and produce the low sow of potential, this will reduce feed and human cost greatly, contribute to the flourish of China's live pig industry.Therefore develop a kind of applicable pig farm, the molecular marker that also accurately can detect sow Oocyte quality without wound is most important.
Summary of the invention
The present invention is directed to above-mentioned Problems existing to make improvements, the serum miRNA proposing a kind of rapid detection sow Oocyte quality marks.
The present invention is achieved by the following technical solutions:
The present invention filters out the microRNA of overexpression from the low ovocyte of potentiality of development by microRNA gene chip, and verified by realtimePCR, final clear and definite miR-23a and Oocyte quality height correlation, can as the marker of rapid detection Oocyte quality.
Because the miR-23a of overexpression can be secreted in serum by the active secretion process of tissue in the low ovocyte of potentiality of development, so the ripe body of selective analysis of the present invention Growth Results low sow serum miR-23a (its sequence is 5 '-AUCACAUUGCCAGGGAUUUCC-3 ', as shown in SEQIDNO:1) expression, Late Cambrian serum miR-23a can apply as rapid detection sow Oocyte quality molecular marker.During embody rule, occur the next morning of the blood sample collection on an empty stomach of quiet vertical reflection sow, adopt RealtimePCR to detect the expression level of miR-23a in serum, if can detect, then this sow should be eliminated.
For achieving the above object, the invention provides a kind of miRNA molecule for rapid detection sow Oocyte quality mark miR-23a, the nucleotide sequence of described molecule marker is as shown in sequence table SEQ IDNO:1, described molecule marker miR-23a is overexpression in potentiality of development difference cumulus oocyte, and is secreted in serum by the active secretion process of tissue; Secretion is used for the rapid detection of sow Oocyte quality to the nucleic acid molecule mark miR-23a in serum.
For achieving the above object, present invention also offers for detecting described molecule marker miR-23a primer pair, nucleotide sequence is as follows:
Forward primer: 5 '-ATCACATTGCCAGGGATTTCC-3 ', as shown in SEQIDNO.2;
Reverse primer sequence is general aptamer primer.
Described primer pair specific amplification goes out the nucleotide sequence shown in SEQIDNO:1.
For achieving the above object, present invention also offers a kind of test kit for rapid detection sow Oocyte quality, comprise above-mentioned primer pair.
Described test kit also comprises 2.5U/ μ lPolyA polysaccharase, reversed transcriptive enzyme, 5 × RT Buffer, removes RNA enzyme water, 2 × qPCR mixed solution, 70% ethanol, serum lysate, chloroform and dehydrated alcohol.
Described test kit specifically composed as follows:
2.5U/ μ lPolyA polysaccharase 20 μ l;
Reversed transcriptive enzyme 20 μ l;
5 × RT Buffer 100 μ l;
Remove RNA enzyme water 1000 μ l;
2 × qPCR mixed solution 2000 μ l;
50um upstream specific primer 20 μ l;
50um lower adaptation primer 20 μ l;
Internal reference upstream primer 20 μ l;
Internal reference downstream primer 20 μ l;
Serum lysate 100ml;
70% ethanol 100ml;
Chloroform 100ml;
Dehydrated alcohol 100ml.
The concrete grammar adopting mentioned reagent box to detect miR-23a is:
(1) extraction of microRNA:
1. get the peripheral blood serum 400 μ l of sow, add 750 μ l lysate MRL, several times, concuss mixes, and at room temperature hatches 5min in piping and druming, and careful supernatant of drawing proceeds in the centrifuge tube of a new RNasefree.
2. add 1mLTrizol reagent, mixing, ambient temperatare puts 10 minutes.Centrifugal 15 minutes of 4 DEG C of 12000rpm.
3. getting supernatant liquor goes in the EP pipe of RNase to 1.5mL, and add 200 μ l chloroforms, thermal agitation mixes 30 seconds, and make aqueous phase and organic fully mixing of being on good terms, room temperature leaves standstill 15 minutes, 4 DEG C of 12000rpm frozen centrifugations 10 minutes.
4. draw upper strata aqueous portion to move to another new EP of RNase that goes and manage, add 0.5mL Virahol, concussion mixing left at room temperature 10 minutes afterwards, 4 DEG C of 12000rpm frozen centrifugations 10 minutes.
5. remove supernatant, add 70% ethanol with precipitated rna composition, and add appropriate DEPC water and dissolved.
(2) detection of miR-23a:
1. RNA enzyme water is gone to supply 25 μ l according to the RNA+ RT Buffer 5 μ l+ reversed transcriptive enzyme 1 μ l+2.5U/ μ l polysaccharase 1 μ l+ obtained.
2. 37 DEG C of water-bath 60min; Be put on ice after 85 DEG C of water-bath 5min, the microRNAcDNA obtained.
3. cDNA2 μ l+2xqPCR mixed solution 10 μ l+ primer (2 μMs) 2 μ l+ universal primer (2 μMs) 2 μ l+DDW4 μ l.
4. denaturation 95 DEG C of 10min → (sex change 15 seconds → annealing 60 DEG C 20 seconds → extend 72 DEG C 30 seconds) 40 circulations, carry out melt curve analysis analysis immediately after end, solubility curve select 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 the Ct value obtained, deduct reference gene, substitute into 2-Δ Δ CT, obtain the expression values of miR-23a in this sow serum.
For achieving the above object, present invention also offers a kind of method of the specific amplification for detecting the ripe body of serum miR-23a, comprise the steps: the extracting first carrying out serum microRNA, then polyA tail is added at poly (A) polysaccharase at its 3' end, under the effect of ThermoScript II, reverse transcription is cDNA, and carries out RealtimePCR amplification.
For achieving the above object, present invention also offers following arbitrary application:
(1) above-mentioned molecule marker is detecting the application in Oocyte quality prediction sow reproductive potential;
(2) above-mentioned primer pair is detecting the application in Oocyte quality prediction sow reproductive potential;
(3) above-mentioned test kit is detecting the application in Oocyte quality prediction sow reproductive potential;
(4) above-mentioned method is detecting the application in Oocyte quality prediction sow reproductive potential.
Beneficial effect of the present invention:
The present invention utilizes the sow ovocyte microRNA express spectra difference that microRNA genechip detection and comparative development potential difference and potentiality of development are high, therefrom filter out candidate microRNA, verified by realtimePCR, find that miR-23a can as the molecular marked compound detecting Oocyte quality.The miR-23a of overexpression can be secreted in serum by the active secretion process of tissue, therefore large sample analysis Growth Results low sow serum miR-23a expression level subsequently, find serum miR-23a be a kind of can the molecular marked compound of rapid detection sow Oocyte quality.This miRNA molecule provided by the invention, as the molecular marker of rapid detection sow Oocyte quality, produces potential to prediction sow significant.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
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.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1 chip method comparative development potential low cumulus oocyte miRNA express spectra change difference
Gather Market pigs ovary from slaughterhouse, ovary transports laboratory back as early as possible in 37 DEG C of stroke-physiological saline solution.The ovarian follicle of Ovarian surface diameter at 2-6mm is extracted with the asepsis injector of No. 20 syringe needles, the liquor folliculi of extraction is slowly injected the centrifuge tube that autoclaving is crossed, make ovocyte natural subsidence 10 minutes, then slowly draw ovarian follicle supernatant liquor with pipettor, will remove in 60mm culture dish that the precipitation after supernatant transfers to containing PBS damping fluid.Under stereoscopic microscope, be divided into two classes to transfer in 1ml centrifuge tube cumulus oocyte with mouth suction pipe ,-70 DEG C of preservations are to be measured.Criteria for classification: the low cumulus oocyte of potentiality of development: oocyte cytoplasm is even, only has individual layer cumulus cell or without cumulus cell bag quilt.Normal group: ovocyte matter is even, has more than 4 layers cumulus cell bag quilts around.
Extract RNA in cumulus oocyte by the following method.
1. cumulus oocyte directly adds Trizol, and add 1mLTrizol solution by 0.1g, put upside down mixing, room temperature leaves standstill 5min;
2. every 1mLTrizol adds 0.2mL chloroform.Cover tightly sample hose lid, firmly rock 15sec, make it fully mix, room temperature leaves standstill the centrifugal 15min of 12000rmp after 5min;
3. upper strata aqueous phase is proceeded in new 1.5mLEP pipe, add equal-volume Virahol, be put in room temperature after mixing and leave standstill 10min, the centrifugal 10min of 12000rmp;
4. abandon supernatant, precipitation is placed on ventilation, dry;
5. 10 μ lDEPC water, 65 DEG C of dissolutions are added.
The RNA of extraction is carried out the expression chip analysis of miRNA, the results are shown in Table 1.Find by table 1, miR-23a has significant difference in the low cumulus oocyte of potentiality of development and Normal group are expressed, the sequence of described miR-23a is as shown in sequence 1, and its sequence is: 5 '-AUCACAUUGCCAGGGAUUUCC-3 ' (SEQIDNO:1).The specific forward direction primer sequence of described miR-23a is: 5 '-ATCACATTGCCAGGGATTTCC-3 ' (SEQIDNO:2); Reverse primer sequence is general aptamer primer.
The change difference of the low cumulus oocyte of table 1 chip analysis potentiality of development and Normal group miRNA express spectra
Embodiment 2 result of real-timePCR technical identification chip
Gather Market pigs ovary from slaughterhouse, ovary transports laboratory back as early as possible in 37 DEG C of stroke-physiological saline solution.The ovarian follicle of Ovarian surface diameter at 2-6mm is extracted with the asepsis injector of No. 20 syringe needles, the liquor folliculi of extraction is slowly injected the centrifuge tube that autoclaving is crossed, make ovocyte natural subsidence 10 minutes, then slowly draw ovarian follicle supernatant liquor with pipettor, will remove in 60mm culture dish that the precipitation after supernatant transfers to containing PBS damping fluid.Under stereoscopic microscope, be divided into two classes to transfer in 1ml centrifuge tube cumulus oocyte with mouth suction pipe ,-70 DEG C of preservations are to be measured.Criteria for classification: the low cumulus oocyte of potentiality of development: oocyte cytoplasm is even, only has individual layer cumulus cell or without cumulus cell bag quilt.Normal group: ovocyte matter is even, has more than 4 layers cumulus cell bag quilts around.
Extract RNA in cumulus oocyte by the following method:
1. cumulus oocyte directly adds Trizol, and add 1mLTrizol solution by 0.1g, put upside down mixing, room temperature leaves standstill 5min;
2. every 1mLTrizol adds 0.2mL chloroform.Cover tightly sample hose lid, firmly rock 15sec, make it fully mix, room temperature leaves standstill the centrifugal 15min of 12000rmp after 5min;
3. upper strata aqueous phase is proceeded in new 1.5mLEP pipe, add equal-volume Virahol, be put in room temperature after mixing and leave standstill 10min, the centrifugal 10min of 12000rmp;
4. abandon supernatant, precipitation is placed on ventilation, dry;
5. 10 μ lDEPC water, 65 DEG C of dissolutions are added.
MicroRNA reverse transcription
Get 2 μ gRNA, at thaw at RT, be placed in rapidly on ice after thawing.Mixed solution is prepared according to table 3.
The reaction system of table 3microRNA reverse transcription
37 DEG C, 60min; 85 DEG C, be put on ice after 5min, the microRNAcDNA obtained can be used for subsequent experimental, or is placed in-20 DEG C of preservations.
Real-timePCR gene expression detection
The cDNA obtained with above-mentioned reverse transcription reaction is template, and the multiple hole of each template-setup of each testing gene three, operate on ice, reaction system preparation is as shown in table 4.
Table 4RealtimePCR reaction system
Reactions steps is set according to two-step approach PCR.Reaction terminates, and according to melting curve and Ct value, analyzes after obtaining the Relative Expression values of goal gene in this template according to formula.Result is as shown in table 2,
Table 2real-timePCR detects the expression level of miR-23a in the low cumulus oocyte of potentiality of development and normal control
| 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 " represents normal control, and " 1 " represents the low cumulus oocyte of potentiality of development.
Carry out statistical study through SPASS software, miR-23a expression level in the low cumulus oocyte of potentiality of development is significantly higher than Normal group (P < 0.05), and result as shown in Figure 1.
Can determine thus, there is significant difference in miR-23a in the low cumulus oocyte of potentiality of development, can as the molecule marker detecting Oocyte quality.
Embodiment 3 expression level of miR-23a in real-timePCR technology for detection Oocyte quality difference sow serum
Serum sample is collected: sow occurs that quiet vertical reflection gathers 10ml blood in short solidifying pipe, 2000 ~ 3000rpm centrifugal 18 ~ 20min the next morning on an empty stomach, and absorption supernatant is in without in RNase centrifuge tube, and 400 μ l packing ,-80 DEG C freezing to be measured.
Research sample selection criteria: continuous two parity litter sizes lower than 5, Body Condition Score (2.5-3.5), the female Landrace of normal health of searching for food is defined as Oocyte quality difference group; To organize parity identical with Oocyte quality difference, and continuous two parity litter size 10-13 heads, Body Condition Score (2.5-3.5), normal healthy female Landrace of searching for food is defined as Normal group.Collect 100 samples altogether, wherein 50 from low group of litter size, and 50 from Normal group.
The extraction of serum microRNA
Step is as follows:
1. get the peripheral blood serum 400 μ l of sow, add 750 μ l lysate MRL, several times, concuss mixes, and at room temperature hatches 5min in piping and druming, and careful supernatant of drawing proceeds in the centrifuge tube of a new RNasefree.
2. add 1mLTrizol reagent, mixing, ambient temperatare puts 10 minutes.Centrifugal 15 minutes of 4 DEG C of 12000rpm.
3. getting supernatant liquor goes in the EP pipe of RNase to 1.5mL, and add 200 μ l chloroforms, thermal agitation mixes 30 seconds, and make aqueous phase and organic fully mixing of being on good terms, room temperature leaves standstill 15 minutes, 4 DEG C of 12000rpm frozen centrifugations 10 minutes.
4. draw upper strata aqueous portion to move to another new EP of RNase that goes and manage, add 0.5mL Virahol, concussion mixing left at room temperature 10 minutes afterwards, 4 DEG C of 12000rpm frozen centrifugations 10 minutes.
5. remove supernatant, add 70% ethanol with precipitated rna composition, and add appropriate DEPC water and dissolved.
MicroRNA reverse transcription
Get 2 μ gRNA, at thaw at RT, be placed in rapidly on ice after thawing.Mixed solution is prepared according to table 3.
The reaction system of table 3microRNA reverse transcription
37 DEG C, 60min; 85 DEG C, be put on ice after 5min, the microRNAcDNA obtained can be used for subsequent experimental, or is placed in-20 DEG C of preservations.
Real-timePCR gene expression detection
The cDNA obtained with above-mentioned reverse transcription reaction is template, and the multiple hole of each template-setup of each testing gene three, operate on ice, reaction system preparation is as shown in table 4.
Table 4RealtimePCR reaction system
Reactions steps is set according to two-step approach PCR.Reaction terminates, and according to melting curve and Ct value, analyzes after obtaining the Relative Expression values of goal gene in this template according to formula.Result is as shown in table 5,
Table 5real-timePCR detects the expression level of miR-23a in normal and the low sow serum of litter size
In table 5, " 0 " represents Normal group (continuous two parity litter size 10-13 heads), and " 1 " represents Oocyte quality difference sow (continuous two parity litter sizes are lower than 5).
Carry out statistical study through spass software, the expression level of miR-23a in Oocyte quality difference group and Normal group has significant difference (p<0.05), and result as shown in Figure 2.
Can determine thus, there is significant difference in miR-23a, can as the molecule marker detecting sow Oocyte quality in Oocyte quality difference group sow serum.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. the mark of the miRNA molecule for a rapid detection sow Oocyte quality miR-23a, it is characterized in that: the nucleotide sequence of described molecule marker is as shown in sequence table SEQ IDNO:1, described molecule marker miR-23a is overexpression in potentiality of development difference cumulus oocyte, and is secreted in serum by the active secretion process of tissue.
2. a kind of miRNA molecule for rapid detection sow Oocyte quality mark miR-23a according to claim 1, is characterized in that: secrete to the rapid detection of the nucleic acid molecule mark miR-23a in serum for sow Oocyte quality.
3. test right requires molecule marker miR-23a primer pair described in 1, it is characterized in that: nucleotide sequence is as follows:
Forward primer: 5 '-ATCACATTGCCAGGGATTTCC-3 ', as shown in SEQIDNO.2;
Reverse primer sequence is general aptamer primer.
4. primer pair according to claim 3, is characterized in that: described primer pair specific amplification goes out the nucleotide sequence shown in SEQIDNO:1.
5. for a test kit for rapid detection sow Oocyte quality, it is characterized in that: comprise claim 3 or primer pair according to claim 4.
6. a kind of test kit for detecting sow Oocyte quality according to claim 5, is characterized in that: also comprise 2.5U/ μ lPolyA polysaccharase, reversed transcriptive enzyme, 5 × RT Buffer, remove RNA enzyme water, 2 × qPCR mixed solution, 70% ethanol, serum lysate, chloroform and dehydrated alcohol.
7. a kind of test kit for detecting sow Oocyte quality according to claim 5, is characterized in that: test kit specifically composed as follows:
2.5U/ μ lPolyA polysaccharase 20 μ l;
Reversed transcriptive enzyme 20 μ l;
5 × RT Buffer 100 μ l;
Remove RNA enzyme water 1000 μ l;
2 × qPCR mixed solution 2000 μ l;
50um upstream specific primer 20 μ l;
50um lower adaptation primer 20 μ l;
Internal reference upstream primer 20 μ l;
Internal reference downstream primer 20 μ l;
Serum lysate 100ml;
70% ethanol 100ml;
Chloroform 100ml;
Dehydrated alcohol 100ml.
8. one kind for detecting the method for the specific amplification of the ripe body of serum miR-23a, it is characterized in that: the extracting comprising the steps: first to carry out serum microRNA, then polyA tail is added at poly (A) polysaccharase at its 3' end, under the effect of ThermoScript II, reverse transcription is cDNA, and carries out RealtimePCR amplification.
9. following arbitrary application:
(1) molecule marker according to claim 1 is detecting the application in Oocyte quality prediction sow reproductive potential;
(2) primer pair described in claim 3 or 4 is detecting the application in Oocyte quality prediction sow reproductive potential;
(3) in claim 5-7, arbitrary described test kit is detecting the application in Oocyte quality prediction sow reproductive potential;
(4) method described in claim 8 is detecting the application in Oocyte quality prediction sow reproductive potential.
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| CN106434912A (en) * | 2016-09-22 | 2017-02-22 | 安徽农业大学 | Applications of microRNA markers relevant to litter size of sow |
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| CN110592232A (en) * | 2019-09-23 | 2019-12-20 | 四川农业大学 | Molecular marker related to pork pH value character and application thereof |
| CN111560443A (en) * | 2020-06-19 | 2020-08-21 | 湖南人文科技学院 | Group of biomolecular markers for screening high-quality porcine oocytes and application thereof |
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