CN105200132A - Heterocephalus glaber VEGFA gene specific detection primer and kit - Google Patents
Heterocephalus glaber VEGFA gene specific detection primer and kit Download PDFInfo
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- CN105200132A CN105200132A CN201510612405.5A CN201510612405A CN105200132A CN 105200132 A CN105200132 A CN 105200132A CN 201510612405 A CN201510612405 A CN 201510612405A CN 105200132 A CN105200132 A CN 105200132A
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention relates to a heterocephalus glaber VEGFA gene specific detection primer and kit and belongs to the field of biotechnological detection. The heterocephalus glaber VEGFA gene detection kit mainly comprises the VEGFA gene Real-time PCR detection primer. The forward primer is shown in the SEQ ID NO:1, and the reverse primer is shown in the SEQ ID NO:2. The invention further provides a heterocephalus glaber VEGFA gene detection method. According to the method, the total mRNA of heterocephalus glaber cells is extracted and subjected to inverse transcription to generate cDNA to serve as a detection sample, detection is conducted with the Real-time PCR method, the mRNA expression level of VEGFA is obtained, and the dynamic change of the mRNA expression level of VEGFA genes can be specifically monitored in a quantitative and qualitative mode by means of the Real-time PCR detection technique. The method is simple and fast and is suitable for application and popularization.
Description
Technical field
The invention belongs to field of biological technology detection, be specifically related to naked mole VEGFA technique of gene detection.
Background technology
Naked mole be a kind of move in for a long time to exchange with ambient atmos have some setbacks, gas concentration lwevel in air is high, and the rodents Mammals in the cave environment of oxygen content extremely low (10% ~ 15%).So it has the ability of extremely strong tolerance low-oxygen environment, it is the good animal model of research lower oxygen concentration resistance mechanism.Research shows, tumor by local hypoxic-ischemic microenvironment has vital role in the generation of tumour, development and treatment opposing.Regional hypoxia microenvironment is one of essential characteristic of noumenal tumour, and hypoxemia microenvironment is not only the initiating agent of tumour generation vicious transformation, its can also inducing tumor cell to radiochemotherapeutic opposing.So the research naked mole being carried out to lower oxygen concentration resistance mechanism is significant for solving the radiotherapy opposing occurred in tumor therapeutic procedure.Research finds, be no matter ontogenetic process or low-oxygen environment stress process in, the generation of blood vessel strictly depends on vascular endothelial growth factor (vascularendothelialgrowthfactor, VEGF) (GeneID:7422 of the VEGFA of people, the GeneID:22339 of the VEGFA of mouse).The laboratory research in earlier stage at the present inventor place also finds, under normal oxygen condition, middle VEGFAmRNA (GeneID:101702403) level of naked mole tissue (brain, lung, the heart, liver, kidney, muscle) of growing up is the hundred times of adult mice, thus points out the composing type high expression level of this gene may adapt to play a significant role in low-oxygen environment naked mole.So be conducive to disclosing its lower oxygen concentration resistance mechanism that may exist to the detection of naked mole VEGF gene expression dose.
At present, due to naked mole breeding cycle long (female naked mole age at sexual maturity 228 days, male naked mole age at sexual maturity 12 months (being 6.5 times and 8.1 times of mouse respectively)), the Gestation period (68 days) of naked mole is 3.2 times of mouse (21 days)), and naked mole belongs to eusociality life Mammals, only has " queen " female naked mole to have the chance of breeding in a colony.Further, wild naked mole moves in the arid area of East Africa Ethiopia, Kenya and Somalia's tropical grassland, is difficult to catch.These all reasons all cause naked mole source quantity relatively less, greatly constrain its applied research.And somatic cell culture technology can solve the problem of research material source difficulty well.The features such as cultured cell in vitro has stronger division, multiplication capacity, and strong adaptability is easily cultivated, and proterties is stable, apply in the middle of current research widely.
At present, the detection of VEGFA genetic expression is mainly carried out to the detection (EisenachPA of expression of gene protein level in the middle of scientific research by westernblotting and ELISA detection kit, etal. (2010) MT1-MMPregulatesVEGF-AexpressionthroughacomplexwithVEGFR-2andSrc.JCellSci.123 (Pt23): 4182-4193.) but need the antibody of application specific in present method, and be not directed to the specific antibodies of naked mole at present, use for other species (mouse, rabbit, the mankind) antibody there is the not enough problem of bonding force, and some cell type is (as skin flbroblast, stellate cells) in VEGFA expressing quantity lower, westernblotting detects and easily can't detect, and unstable result (see Fig. 1).In addition, detect in the process of mrna expression at Real-timePCR, because the specificity designing primer exists very big-difference, thus causing PCR to react the easily non-specific fragment of generation or primer dimer, there are multiple peaks phenomenon (see Fig. 4) in the solubility curve namely reacted.
There is no specific detection primer and detection kit that bibliographical information relates to naked mole VEGFA gene at present.
Summary of the invention
In order to solve the deficiency of above-mentioned detection technique, the invention provides primer and the detection kit of VEGFA gene Real-timePCR detection.
A first aspect of the present invention, provide a kind of naked mole VEGFA gene specific to detect primer, VEGFA gene Real-timePCR detects primer (2OD) and is:
Forward primer: 5 '-TGCCCTTGGTGGGGTTTG-3 ' (SEQIDNO:1),
Reverse primer: 5 '-CGAGACGCTGGTGGACATC-3 ' (SEQIDNO:2).
A second aspect of the present invention, provides a kind of detection kit of naked mole VEGFA gene, comprises VEGFA gene Real-timePCR and detects primer (2OD):
Forward primer: as shown in SEQIDNO:1,
Reverse primer: as shown in SEQIDNO:2.
Above-mentioned naked mole VEGFA gene specific detection kit, also comprises:
PremixExTaq
TM5000μl，ROXReferenceDye(50×)200μl。
Preferably, the detection kit of a kind of naked mole VEGFA gene of the present invention, comprising:
Forward primer: as shown in SEQIDNO:1,2OD;
Reverse primer: as shown in SEQIDNO:2,2OD;
PremixExTaq
TM，5000μl；
ROXReferenceDye(50×)，200μl；
Sterile purified water appropriate (preferably 5000 μ l, each test kit can do 1000 PCR reactions).
A third aspect of the present invention provides a kind of detection method of naked mole VEGFA gene, and described detection method utilizes above-mentioned specific detection primer; Or utilizing above-mentioned detection kit, described detection method comprises the steps:
Extract total mRNA of cell, then reverse transcription becomes cDNA, and with this cDNA for template, utilize the Real-timePCR of VEGFA gene to detect primer and carry out quantitative fluorescent PCR reaction, primer sequence is as follows:
Forward primer: 5 '-TGCCCTTGGTGGGGTTTG-3 ' (SEQIDNO:1);
Reverse primer: 5 '-CGAGACGCTGGTGGACATC-3 ' (SEQIDNO:2).
The method that above-mentioned naked mole VEGFA gene specific detects, concrete detecting step is:
1) total serum IgE in cell or tissue is extracted:
Adopt TRIZOL method to extract total serum IgE in cell or tissue, and adopt the reverse transcription of Reverse Transcription box to be cDNA (Reverse Transcription box, Tian Gen biochemical technology company)
2) Real-timePCR detection reaction system 10 μ l is prepared:
By step 1) in get 1 μ l after cDNA solution dilution 10 times,
premixExTaq
tM5 μ l, PCR forward primer (PCRForwardprimer) dilution is get 0.2 μ l after 10 μMs of concentration, PCR reverse primer (PCRreverseprimer) dilution is get 0.2 μ l after 10 μMs of concentration, ROXReferenceDye (50 ×) 0.2 μ l, sterile purified water 3.4 μ l.Negative control is set.
3) carry out PCR reaction, response procedures is as follows:
Holding stage (Holdingstage) 95 DEG C of 30s; Cycle stage (cyclingstage) 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage (Meltcurvestage) 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.
Described TRIZOL method is: get normal oxygen condition (20%O
2, 5%CO
2) naked 3 parts, the mole skin flbroblast sample cultivated, be labeled as 1,2,3, get hypoxemia culture condition (3%O
2, 92%N
2, 5%CO
2) naked 3 parts, the mole skin flbroblast sample cultivated, be labeled as 4,5,6.(can see ChomczynskiP (1993) Areagentforthesingle-stepsimultaneousisolationofRNA, DNAandproteinsfromcellandtissuesamples.Biotechniques15:5 32-534,536-537.)
Technique effect of the present invention is as follows:
Primer used detects for the mRNA level in-site of VEGFA, extract total mRNA of naked mole cell, and reverse transcription is that cDNA is as detection sample, Real-timePCR method is used to detect, draw the mrna expression level of VEGFA, utilize Real-timePCR detection technique can specifically quantitatively and the dynamic change of qualitative monitoring VEGFA mrna expression, simple and fast, be applicable to promoting the use of.As judging the basis for estimation that naked mole VEGFA genetic expression changes, there is good specificity and practicality.
Accompanying drawing explanation
Fig. 1 is for using anti-mouse VEGFA antibody, and Westernblot detects VEGFA protein expression design sketch in naked mole skin flbroblast, and (wherein sample 1 is (O under normal oxygen condition
2volume fraction 20%) cell protein sample, arrange two repetitions, sample 2 is (O under hypoxia condition
2volume fraction 3%) cell protein sample, two repetitions are set);
Fig. 2 is the solubility curve that the Real-timePCR of embodiment 1 detects;
Fig. 3 is the amplification curve that the Real-timePCR of embodiment 1 detects.
Fig. 4 is (a, b, solubility curves c) detected with primer sequence (SEQIDNO:1, SEQIDNO:2) (d) Real-timePCR in the present invention of other three pairs of primers in embodiment 2.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further illustrated.
Embodiment 1:
Extract total mRNA of cell, then reverse transcription becomes cDNA, and with this cDNA for template, the Real-timePCR of application VEGFA gene detects primer and test kit, and primer sequence is as follows:
Forward primer: 5 '-TGCCCTTGGTGGGGTTTG-3 ' (SEQIDNO:1)
Reverse primer: 5 '-CGAGACGCTGGTGGACATC-3 ' (SEQIDNO:2), carries out quantitative fluorescent PCR reaction.
Detecting step:
1) cell total rna is extracted:
Wherein get the normal oxygen condition (O of 20% volume fraction
2, 5% volume fraction CO
2) naked 3 parts, the mole skin flbroblast sample cultivated, be labeled as 1,2,3, get the hypoxemia culture condition (O of 3% volume fraction
2, 92% volume fraction N
2, 5% volume fraction CO
2) naked 3 parts, the mole skin flbroblast sample cultivated, be labeled as 4,5,6, extract cell total rna, and reverse transcription be cDNA (Reverse Transcription box, Tian Gen biochemical technology company)
2) Real-timePCR detection reaction system 10 μ l is prepared:
By step 1) in get 1 μ l after cDNA solution dilution 10 times,
premixExTaq
tM5 μ l, PCR forward primer (PCRForwardprimer) (10 μMs) 0.2 μ l, PCR reverse primer (PCRreverseprimer) (10 μMs) 0.2 μ l, ROXReferenceDye (50 ×) 0.2 μ l, sterile purified water 3.4 μ l.Negative control is set.
3) react, temperature of reaction is as follows:
Holding stage (Holdingstage) 95 DEG C of 30s; Cycle stage (cyclingstage) 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage (Meltcurvestage) 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.
The solubility curve of Fig. 2 shows the specificity (in figure the normal oxygen group of curve representation (1,2,3) and hypoxemia treatment group (4,5,6) solubility curve design sketch) that VEGFA gene primer increases; The amplification curve of Fig. 3 represents the real-time fluorescent PCR amplification curve of normal oxygen group (1,2,3) and hypoxemia treatment group (4,5,6).
As in the solubility curve figure of Fig. 2 example 1, all there is single peak value in all sample PCR reactions of detection, solvent temperature Tm value is 85.6 DEG C, illustrates that quantitative fluorescent PCR reaction has good specificity.In the amplification curve of Fig. 3, ordinate zou Δ Rn refers to standard indicator signal value and deducts baseline signal value, and X-coordinate represents the circulation number of turns, as seen from the figure, what the Δ Rn value detecting all samples all counted up at PCR circulating ring advances into plateau, PCR reaction is described effectively.
Embodiment 2
Design other three pairs of primers according to the mRNA sequence information of mole VEGFA naked on GeneBank simultaneously:
a：
Forward primer 5 '-GGGGAGATCGCTCCTTTGC-3 ' (SEQIDNO:3), reverse primer 5 '-GCTCGCTCTTCCTCGACTTCT-3 ' (SEQIDNO:4);
b：
Forward primer 5 '-CAGATCATGCGGATCAAACCC-3 ' (SEQIDNO:5), reverse primer 5 '-TCTGCTGTGCTGGAGGAAACT-3 ' (SEQIDNO:6);
C: forward primer 5 '-TCCGCAGACGTGTAAATGCT-3 ' (SEQIDNO:7), reverse primer 3 '-TTCGTTTAACTCAAGCTGCCTC-3 ' (SEQIDNO:8),
Extract the total mRNA of cell, then reverse transcription is cDNA, together carries out Real-timePCR reaction with the primer sequence (SEQIDNO:1, SEQIDNO:2) in the present invention.Detecting step:
1) cell total rna is extracted: get the normal oxygen condition (O of 20% volume fraction
2, 5% volume fraction CO
2) the naked mole skin flbroblast sample cultivated and the hypoxemia culture condition (O of 3% volume fraction
2, 92% volume fraction N
2, 5% volume fraction CO
2) the naked mole skin flbroblast sample cultivated, extract cell total rna, and reverse transcription is cDNA (Reverse Transcription box, Tian Gen biochemical technology company)
2) Real-timePCR detection reaction system 10 μ l is prepared:
By step 1) in get 1 μ l after cDNA solution dilution 10 times,
premixExTaq
tM5 μ l, PCR forward primer (PCRForwardprimer) (10 μMs) 0.2 μ l, PCR reverse primer (PCRreverseprimer) (10 μMs) 0.2 μ l, ROXReferenceDye (50 ×) 0.2 μ l, sterile purified water 3.4 μ l.Negative control is set.
3) react, temperature of reaction is as follows:
Holding stage (Holdingstage) 95 DEG C of 30s; Cycle stage (cyclingstage) 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage (Meltcurvestage) 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.
The solubility curve of Fig. 4 represents three pairs of primers (a, b, solubility curve design sketch c) reacted with the PCR of the primer (d) in invention respectively
As in the solubility curve figure of Fig. 4 example 2, (all there is multiple peak value in the solubility curve of a, b, c) PCR reaction to the three pairs of primers detected, all there is no unique solvent temperature Tm value, illustrate that the quantitative fluorescent PCR reaction of primer does not have specificity, there is nonspecific amplification, result can not be used for the mrna expression level judging gene.And the solubility curve (d) of primer sequence (SEQIDNO:1, SEQIDNO:2) in the present invention has single peak value, illustrate that PCR reaction possesses good specificity, may be used for the expression level change judging VEGFA gene mRNA, reliable results.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (6)
1. naked mole VEGFA gene specific detects a primer, it is characterized in that, described detection primer is that VEGFA gene Real-timePCR detects primer:
Forward primer as shown in SEQIDNO:1,
Reverse primer is as shown in SEQIDNO:2.
2. a detection kit for naked mole VEGFA gene, is characterized in that, described detection kit comprises VEGFA gene Real-timePCR and detects primer:
Forward primer as shown in SEQIDNO:1,
Reverse primer is as shown in SEQIDNO:2.
3. the detection kit of naked mole VEGFA gene according to claim 2, it is characterized in that, described detection kit also comprises:
PremixExTaq
TM；
50×ROXReferenceDye。
4. the detection kit of naked mole VEGFA gene according to claim 2, it is characterized in that, described detection kit comprises:
Forward primer as shown in SEQIDNO:1,2OD;
Reverse primer as shown in SEQIDNO:2,2OD;
PremixExTaq
TM，5000μl；
50×ROXReferenceDye，200μl；
Sterile purified water, in right amount.
5. a detection method for naked mole VEGFA gene, it is characterized in that, described detection method comprises the steps:
Extract total mRNA of cell, then reverse transcription becomes cDNA, and with this cDNA for template, utilize the Real-timePCR of VEGFA gene to detect primer and carry out quantitative fluorescent PCR reaction, primer sequence is as follows:
Forward primer is as shown in SEQIDNO:1;
Reverse primer is as shown in SEQIDNO:2.
6. the detection method of naked mole VEGFA gene according to claim 5, it is characterized in that, concrete detecting step is:
A. total serum IgE in cell or tissue is extracted:
Adopt TRIZOL method to extract total serum IgE in cell or tissue, and adopt the reverse transcription of Reverse Transcription box to be cDNA;
B. Real-timePCR detection reaction system 10 μ l is prepared:
1 μ l is got by after cDNA solution dilution in steps A 10 times,
premixExTaq
tM5 μ l, the dilution of PCR forward primer is get 0.2 μ l after 10 μMs of concentration, and the dilution of PCR reverse primer is get 0.2 μ l, 50 × ROXReferenceDye0.2 μ l after 10 μMs of concentration, sterile purified water 3.4 μ l; Negative control is set;
C. carry out PCR reaction, response procedures is as follows:
Holding stage 95 DEG C of 30s; Cycle stage 95 DEG C of 5s, 60 DEG C of 34s, 72 DEG C of 30s, 40 circulations; Solubility curve stage 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505863A (en) * | 2016-01-05 | 2016-04-20 | 中国人民解放军第二军医大学 | Culture method for heterocephalus glaber cardiac muscle cell |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011107819A1 (en) * | 2010-03-01 | 2011-09-09 | Adelbio | Methods for predicting outcome of breast cancer, and/or risk of relapse, response or survival of a patient suffering therefrom |
| WO2011107939A1 (en) * | 2010-03-01 | 2011-09-09 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Methods of predicting efficacy of an anti-vegfa treatment for solid tumors |
| WO2014089055A1 (en) * | 2012-12-03 | 2014-06-12 | Aveo Pharmaceuticals, Inc. | Tivozanib response prediction |
Non-Patent Citations (3)
| Title |
|---|
| NCBI REFERENCE SEQUENCE:XM_013072755.1: "PREDICTED: Heterocephalus glaber vascular endothelial growth factor A(Vegfa), partial mRNA", 《GENBANK》 * |
| 肖邦 等: "裸鼹鼠不同组织中低氧相关基因的表达", 《实验动物与比较医学》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505863A (en) * | 2016-01-05 | 2016-04-20 | 中国人民解放军第二军医大学 | Culture method for heterocephalus glaber cardiac muscle cell |
| CN105505863B (en) * | 2016-01-05 | 2019-03-22 | 中国人民解放军第二军医大学 | A kind of cultural method of naked mole cardiac muscle cell |
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Application publication date: 20151230 |