CN109112182A - The method of the single oocyte gene quantitative expression of one boar - Google Patents
The method of the single oocyte gene quantitative expression of one boar Download PDFInfo
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- CN109112182A CN109112182A CN201811027188.3A CN201811027188A CN109112182A CN 109112182 A CN109112182 A CN 109112182A CN 201811027188 A CN201811027188 A CN 201811027188A CN 109112182 A CN109112182 A CN 109112182A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses the methods of the single oocyte gene quantitative expression of a boar, pass through S1: the preparation in primer pond, S2: configuration, the S3:PCR polymerase chain reaction, S4 of reverse transcription reaction system: template DNA dilution, S5:qPCR quantitative polyase chain reaction, to realize the gene quantification expression of the single egg mother cell of pig.The present invention is realized RNA extraction purification, reverse transcription and PCR reaction and completed in same pipe, be not required to operation bidirectional, had and reduce test error, reduce pollution, improve the advantages such as sensitivity using unicellular quantitative PCR；Quantitative PCR is carried out using individual cells, sample requirements is greatly reduced, shortens time and efforts, reduce cost, while also solving the limitation that quantitative PCR technique is applied in precious sample.The method of the present invention, easy to operate, at low cost, error is small, and accuracy is high, and effect is good.
Description
Technical field
The present invention relates to cell biology, the methods of the single oocyte gene quantitative expression of a specially boar.
Technical background
Now, the development of animal embryo engineering technology is greatly promoted animal husbandry and biomedical progress, utilizes it
Domestic animal kind can be improved, cultivate transgenosis new varieties, preparation human disease model, production pharmaceutical protein, research embryonic development
Mechanism etc..And pig is the most important thing of embryo biologist and geneticist's research, because of the physiology of pig and anatomical structure and people
Class has very big similarity.
In Animal husbandry production, low fertility of sow is influence one of production efficiency and the key factor of economic benefit, and
Body early embryo death is considered as one of the main reason for causing fertility of sow to decline, about 30% embryonic death occur by
Essence it is oval to embryo nidation this period of time in.It therefore, is always livestock thremmatology for the research of early embryonic development
Research hotspot.To early embryonic development research conventional method include RNA interference, Western blotting, immunofluorescence dyeing and
Quantitative PCR technique.Wherein quantitative PCR technique can be used for the quantitative analysis of DNA or RNA, Gene expression differential display etc., be modern
The important and essential link of animal embryo biotechnology.
Traditional quantitative PCR technique needs to carry out cell and receives sample, the extraction of total serum IgE, reverse transcription and last Step One
The quantitative analysis of instrument.Traditional qPCR operating procedure is more, and required time is long, increases and occurs sample pollution in operating process
Possibility, and the sample needed for it is relatively more, and tens of pieces thin needed for a quantitative PCR, which limits this technologies in preciousness
Application in sample.
Summary of the invention
It is an object of the invention to: the method for the single oocyte gene quantitative expression of a boar is provided, more than solving
Defect.
To achieve the goals above, the invention provides the following technical scheme:
The method of the single oocyte gene quantitative expression of one boar, comprising the following steps:
The preparation of S1:Assay Pool (primer pond)
By OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1 α 1, ID2, STELLA, CARML, INO80,
P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC, CDX2 totally 25 bases
The amplimer of cause, is mixed in proportion, and primer pond is made, and the respective final concentration of the amplimer of 25 genes is
0.1μM；
The configuration of S2:RT-PreAmp Master Mix (reverse transcription reaction system)
In the centrifuge tube of 4 free nucleic acids, configuration RT-PreAmp Master Mix (reverse transcription reaction system) 5.0 μ
L, and be put on ice for for use, then taking the good porcine oocytes of form, being washed three times with DPBS Du Shi phosphate buffer,
An egg mother cell is respectively added into four centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and places 2min, last 3000rpm condition
Lower centrifugation 2min；
S3:PCR (polymerase chain reaction)
Centrifuge tube after centrifugation is immediately placed in PCR instrument and is reacted, reaction step and control condition are respectively as follows: reverse
Record, 50 DEG C, 60min, 1 circulation；Initial denaturation, 95 DEG C, 3min, 1 circulation；Denaturation, 95 DEG C, 15s, 0 circulation；It anneals, prolong
It stretches, 60 DEG C, 15min, 17 circulations；Maintain 4 DEG C for use；
S4: template DNA dilution
By PCR stand-by template DNA after reaction, it is diluted, is vortexed mixed according to 800~1200 extension rate
It is even, it is centrifuged 2min under the conditions of 3000rpm, then saves backup for -20 DEG C；
S5:qPCR (quantitative polyase chain reaction)
Spare template DNA is centrifuged using dilution and configures qPCR reaction system, is then centrifuged 2min under the conditions of 300rpm,
It is finally immediately placed in qPCR instrument and is reacted, to realize the gene quantification expression of the single egg mother cell of pig.
Preferably, in parts by weight, the component of the RT-PreAmp Master Mix (reverse transcription reaction system) includes:
2~3 parts of 2 × Reaction Mix (reaction mixture), 0.2~0.8 part of Assay Pool (primer pond), RT/Taq enzyme 0.05~
0.15 part, 1.5~2.5 parts of Nuclease free water (water of free nucleic acid).
Preferably, in parts by weight, the component of the qPCR reaction system, comprising:qPCR SYBR Green
9~13 parts of Master Mix, 0.5~2 part of Assay Pool (primer pond), dilution after 0.5~2 part of template DNA,
6~9 parts of Nuclease free water (water of free nucleic acid).
Preferably, the qPCR reaction step and control condition are respectively as follows: step 1: initial denaturation, 95 DEG C, 5min；Step
Two: circular response, 40 recycle, first 95 DEG C, 10s, then 60 DEG C, 30s, last 95 DEG C, 15s；Step 3: solubility curve, 40
A circulation, first 60 DEG C, 60s, then 95 DEG C, 15s.
Preferably, in parts by weight, the component of the RT-PreAmp Master Mix (reverse transcription reaction system) includes:
2.5 parts of 2 × Reaction Mix (reaction mixture), 0.5 part of Assay Pool primer pond, 0.1 part of RT/Taq enzyme, Nuclease
1.9 parts of free water (water of free nucleic acid).
Preferably, in parts by weight, the component of the qPCR reaction system, comprising:qPCR SYBR Green
1 part of template DNA, Nuclease free after 10.4 parts of Master Mix, 1 part of Assay Pool (primer pond), dilution
7.6 parts of water (water of free nucleic acid).
The beneficial effects of the present invention are:
The method of the single oocyte gene quantitative expression of a boar of the invention, only needs template using unicellular quantitative PCR
The amplification of DNA and two step of quantitative analysis of Step One instrument realize RNA extraction purification, reverse transcription and PCR reaction same
It is completed in one pipe, is not required to additional operation, had and reduce test error, reduce pollution, improve the advantages such as sensitivity.Using single
Cell carries out the demand that quantitative PCR greatly reduces sample, shortens the time and efforts of investment, and reduce cost,
Also solves the limitation that quantitative PCR technique is applied in precious sample simultaneously.The method of the present invention, it is easy to operate, it is at low cost, accidentally
Difference is small, and accuracy is high, and effect is good.
Specific embodiment
In order to facilitate the understanding of those skilled in the art, the present invention is further illustrated combined with specific embodiments below.
Embodiment 1:
The method of the single oocyte gene quantitative expression of one boar, comprising the following steps:
The preparation of S1:Assay Pool (primer pond)
By OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1 α 1, ID2, STELLA, CARML, INO80,
P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC, CDX2 totally 25 bases
The amplimer of cause, is mixed in proportion, and primer pond is made, and the respective final concentration of the amplimer of 25 genes is
0.1μM。
The configuration of S2:RT-PreAmp Master Mix (reverse transcription reaction system)
In the centrifuge tube of 4 free nucleic acids, configuration RT-PreAmp Master Mix (reverse transcription reaction system) 5.0 μ
L, and be put on ice for for use, then taking the good porcine oocytes of form, being washed three times with DPBS Du Shi phosphate buffer,
An egg mother cell is respectively added into four centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and places 2min, last 3000rpm condition
Lower centrifugation 2min；
Wherein, in parts by weight, the component of RT-PreAmp Master Mix (reverse transcription reaction system) include: 2 ×
2 parts of Reaction Mix (reaction mixture), 0.2 part of Assay Pool (primer pond), 0.15 part of RT/Taq enzyme, Nuclease
2.5 parts of free water (water of free nucleic acid).
S3:PCR (polymerase chain reaction)
Centrifuge tube after centrifugation is immediately placed in PCR instrument and is reacted, reaction step and control condition are respectively as follows: reverse
Record, 50 DEG C, 60min, 1 circulation；Initial denaturation, 95 DEG C, 3min, 1 circulation；Denaturation, 95 DEG C, 15s, 0 circulation；It anneals, prolong
It stretches, 60 DEG C, 15min, 17 circulations；Maintain 4 DEG C for use.
S4: template DNA dilution
By PCR stand-by template DNA after reaction, it is diluted, is vortexed mixed according to 800~1200 extension rate
It is even, it is centrifuged 2min under the conditions of 3000rpm, then saves backup for -20 DEG C.
S5:qPCR (quantitative polyase chain reaction)
Spare template DNA is centrifuged using dilution and configures qPCR reaction system, is then centrifuged 2min under the conditions of 300rpm,
It is finally immediately placed in qPCR instrument and is reacted, to realize the gene quantification expression of the single egg mother cell of pig.
Wherein, in parts by weight, the component of qPCR reaction system, comprising:qPCR SYBR Green Master
0.5 part of template DNA, Nuclease free water after 9 parts of Mix, 2 parts of Assay Pool (primer pond), dilution is (seedless
The water of acid) 9 parts.
Wherein, qPCR reaction step and control condition are respectively as follows: step 1: initial denaturation, 95 DEG C, 5min；Step 2: circulation
Reaction, 40 recycle, first 95 DEG C, 10s, then 60 DEG C, 30s, last 95 DEG C, 15s；Step 3: solubility curve, 40 circulations,
First 60 DEG C, 60s, then 95 DEG C, 15s.
Embodiment 2:
The method of the single oocyte gene quantitative expression of one boar, comprising the following steps:
The preparation of S1:Assay Pool (primer pond)
By OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1 α 1, ID2, STELLA, CARML, INO80,
P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC, CDX2 totally 25 bases
The amplimer of cause, is mixed in proportion, and primer pond is made, and the respective final concentration of the amplimer of 25 genes is
0.1μM。
The configuration of S2:RT-PreAmp Master Mix (reverse transcription reaction system)
In the centrifuge tube of 4 free nucleic acids, configuration RT-PreAmp Master Mix (reverse transcription reaction system) 5.0 μ
L, and be put on ice for for use, then taking the good porcine oocytes of form, being washed three times with DPBS Du Shi phosphate buffer,
An egg mother cell is respectively added into four centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and places 2min, last 3000rpm condition
Lower centrifugation 2min；
Wherein, in parts by weight, the component of RT-PreAmp Master Mix (reverse transcription reaction system) include: 2 ×
3 parts of Reaction Mix (reaction mixture), 0.8 part of Assay Pool (primer pond), 0.05 part of RT/Taq enzyme, Nuclease
1.5 parts of free water (water of free nucleic acid).
S3:PCR (polymerase chain reaction)
Centrifuge tube after centrifugation is immediately placed in PCR instrument and is reacted, reaction step and control condition are respectively as follows: reverse
Record, 50 DEG C, 60min, 1 circulation；Initial denaturation, 95 DEG C, 3min, 1 circulation；Denaturation, 95 DEG C, 15s, 0 circulation；It anneals, prolong
It stretches, 60 DEG C, 15min, 17 circulations；Maintain 4 DEG C for use.
S4: template DNA dilution
By PCR stand-by template DNA after reaction, it is diluted, is vortexed mixed according to 800~1200 extension rate
It is even, it is centrifuged 2min under the conditions of 3000rpm, then saves backup for -20 DEG C.
S5:qPCR (quantitative polyase chain reaction)
Spare template DNA is centrifuged using dilution and configures qPCR reaction system, is then centrifuged 2min under the conditions of 300rpm,
It is finally immediately placed in qPCR instrument and is reacted, to realize the gene quantification expression of the single egg mother cell of pig.
Wherein, in parts by weight, the component of qPCR reaction system, comprising:qPCR SYBR Green Master
2 parts of template DNA, Nuclease free water after 13 parts of Mix, 0.5 part of Assay Pool (primer pond), dilution is (seedless
The water of acid) 6 parts.
Wherein, qPCR reaction step and control condition are respectively as follows: step 1: initial denaturation, 95 DEG C, 5min；Step 2: circulation
Reaction, 40 recycle, first 95 DEG C, 10s, then 60 DEG C, 30s, last 95 DEG C, 15s；Step 3: solubility curve, 40 circulations,
First 60 DEG C, 60s, then 95 DEG C, 15s.
Embodiment 3:
The method of the single oocyte gene quantitative expression of one boar, comprising the following steps:
The preparation of S1:Assay Pool (primer pond)
By OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1 α 1, ID2, STELLA, CARML, INO80,
P21, P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC, CDX2 totally 25 bases
The amplimer of cause, is mixed in proportion, and primer pond is made, and the respective final concentration of the amplimer of 25 genes is
0.1μM。
The configuration of S2:RT-PreAmp Master Mix (reverse transcription reaction system)
In the centrifuge tube of 4 free nucleic acids, configuration RT-PreAmp Master Mix (reverse transcription reaction system) 5.0 μ
L, and be put on ice for for use, then taking the good porcine oocytes of form, washing three with DPBS (Du Shi phosphate buffer)
It is secondary, an egg mother cell is respectively added into four centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and places 2min, last 3000rpm item
2min is centrifuged under part；
Wherein, in parts by weight, the component of RT-PreAmp Master Mix (reverse transcription reaction system) include: 2 ×
2.5 parts of Reaction Mix (reaction mixture), 0.5 part of Assay Pool (primer pond), 0.1 part of RT/Taq enzyme, Nuclease
1.9 parts of free water (water of free nucleic acid).
S3:PCR (polymerase chain reaction)
Centrifuge tube after centrifugation is immediately placed in PCR instrument and is reacted, reaction step and control condition are respectively as follows: reverse
Record, 50 DEG C, 60min, 1 circulation；Initial denaturation, 95 DEG C, 3min, 1 circulation；Denaturation, 95 DEG C, 15s, 0 circulation；It anneals, prolong
It stretches, 60 DEG C, 15min, 17 circulations；Maintain 4 DEG C for use.
S4: template DNA dilution
By PCR stand-by template DNA after reaction, it is diluted, is vortexed mixed according to 800~1200 extension rate
It is even, it is centrifuged 2min under the conditions of 3000rpm, then saves backup for -20 DEG C.
S5:qPCR (quantitative polyase chain reaction)
Spare template DNA is centrifuged using dilution and configures qPCR reaction system, is then centrifuged 2min under the conditions of 300rpm,
It is finally immediately placed in qPCR instrument and is reacted, to realize the gene quantification expression of the single egg mother cell of pig.
Wherein, in parts by weight, the component of qPCR reaction system, comprising:qPCR SYBR Green Master
1 part of template DNA, Nuclease free water after 10.4 parts of Mix, 1 part of Assay Pool (primer pond), dilution is (seedless
The water of acid) 7.6 parts.
Wherein, qPCR reaction step and control condition are respectively as follows: step 1: initial denaturation, 95 DEG C, 5min；Step 2: circulation
Reaction, 40 recycle, first 95 DEG C, 10s, then 60 DEG C, 30s, last 95 DEG C, 15s；Step 3: solubility curve, 40 circulations,
First 60 DEG C, 60s, then 95 DEG C, 15s.
Above-mentioned is that invention is exemplarily described, it is clear that present invention specific implementation is not subject to the restrictions described above,
As long as using this insubstantial improvement that the inventive concept and technical scheme of the present invention carry out, or the not improved structure by invention
Think and technical solution directly applies to other occasions, it is within the scope of the present invention.
Claims (6)
1. the method for the single oocyte gene quantitative expression of a boar, which comprises the following steps:
The preparation of S1:Assay Pool (primer pond)
By OCT4, NANOG, SOX2, TEL1, TEL2, TEL3, WDR5, EF1 α 1, ID2, STELLA, CARML, INO80, P21,
P27, P53, P57, GAPDH, H2A, METTL3, METTL14, WTAP, ALKBH5, LIN28, L-MYC, CDX2 totally 25 genes
Amplimer is mixed in proportion, primer pond is made, the respective final concentration of the amplimer of 25 genes is 0.1 μ
M；
The configuration of S2:RT-PreAmp Master Mix (reverse transcription reaction system)
In the centrifuge tube of 4 free nucleic acids, configuration RT-PreAmp Master Mix (reverse transcription reaction system) 5.0 μ l, and
It is put on ice for stand-by, then takes the good porcine oocytes of form, with DPBS (Du Shi phosphate buffer washs three times), to
An egg mother cell is respectively added in four centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and places 2min, under the conditions of last 3000rpm
It is centrifuged 2min；
S3:PCR (polymerase chain reaction)
Centrifuge tube after centrifugation is immediately placed in PCR instrument and is reacted, reaction step and control condition are respectively as follows: reverse transcription,
50 DEG C, 60min, 1 circulation；Initial denaturation, 95 DEG C, 3min, 1 circulation；Denaturation, 95 DEG C, 15s, 0 circulation；Annealing extends,
60 DEG C, 15min, 17 circulations；Maintain 4 DEG C for use；
S4: template DNA dilution
By PCR stand-by template DNA after reaction, it is diluted according to 800~1200 extension rate, is vortexed and mixes,
It is centrifuged 2min under the conditions of 3000rpm, then saves backup for -20 DEG C；
S5:qPCR (quantitative polyase chain reaction)
Spare template DNA is centrifuged using dilution and configures qPCR reaction system, 2min is then centrifuged under the conditions of 300rpm, finally
It is immediately placed in qPCR instrument and is reacted, to realize the gene quantification expression of the single egg mother cell of pig.
2. the method for the single oocyte gene quantitative expression of a boar according to claim 1, which is characterized in that with weight
Part meter is measured, the component of the RT-PreAmp Master Mix (reverse transcription reaction system) includes: 2 × Reaction Mix (anti-
Answer mixture) 2~3 parts, 0.2~0.8 part of Assay Pool (primer pond), 0.05~0.15 part of RT/Taq enzyme, Nuclease
1.5~2.5 parts of free water (water of free nucleic acid).
3. the method for the single oocyte gene quantitative expression of a boar according to claim 1, which is characterized in that with weight
Measure part meter, the component of the qPCR reaction system, comprising:9~13 parts of qPCR SYBR Green Master Mix,
0.5~2 part of template DNA, Nuclease free water after 0.5~2 part of Assay Pool (primer pond), dilution is (seedless
The water of acid) 6~9 parts.
4. the method for the single oocyte gene quantitative expression of a boar according to claim 1, which is characterized in that described
QPCR reaction step and control condition are respectively as follows: step 1: initial denaturation, 95 DEG C, 5min；Step 2: circular response, 40 are followed
Ring, first 95 DEG C, 10s, then 60 DEG C, 30s, last 95 DEG C, 15s；Step 3: solubility curve, 40 circulations, first 60 DEG C, 60s,
Then 95 DEG C, 15s.
5. the method for the single oocyte gene quantitative expression of a boar according to claim 1, which is characterized in that with weight
Part meter is measured, the component of the RT-PreAmp Master Mix (reverse transcription reaction system) includes: 2 × Reaction Mix (anti-
Answer mixture) 2.5 parts, 0.5 part of Assay Pool primer pond, 0.1 part of RT/Taq enzyme, Nuclease free water (free nucleic acid
Water) 1.9 parts.
6. the method for the single oocyte gene quantitative expression of a boar according to claim 1, which is characterized in that with weight
Measure part meter, the component of the qPCR reaction system, comprising:10.4 parts of qPCR SYBR Green Master Mix,
1 part of template DNA, Nuclease free water (water of free nucleic acid) 7.6 after 1 part of Assay Pool (primer pond), dilution
Part.
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| US20110293580A1 (en) * | 1999-10-28 | 2011-12-01 | Advanced Cell Technology, Inc. | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
| CN105705659A (en) * | 2013-08-28 | 2016-06-22 | 赛卢拉研究公司 | Massively parallel single cell analysis |
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