CN107858431A - Primer sets, kit and method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression - Google Patents

Primer sets, kit and method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Download PDF

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CN107858431A
CN107858431A CN201711220736.XA CN201711220736A CN107858431A CN 107858431 A CN107858431 A CN 107858431A CN 201711220736 A CN201711220736 A CN 201711220736A CN 107858431 A CN107858431 A CN 107858431A
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vegfr1
vegfr2
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sense primers
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Guangdong Great Medical Laboratory Co Ltd
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Abstract

The present invention relates to a kind of primer sets for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression.The primer sets include being directed to the specific primer and probe of VEGFR1 genes, VEGFR2 genes and reference gene Actin respectively.The present invention relates to a kind of kit for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression.The kit includes PCR reaction solutions, and the PCR reaction solutions include detection VEGFR1 genes, VEGFR2 genes and reference gene Actin specific primer and probe, in addition to PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.The invention further relates to a kind of method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression.Kit provided by the invention and its detection method have the advantages that simple to operate, detection time is short, detection sensitivity is high and specific good, are particularly suitable for the popularization and application in clinical examination works.

Description

Primer for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Group, kit and method
Technical field
The present invention relates to vitro diagnostic techniques field, particularly, is related to one kind and is used for cancer target medicine phases correlation gene Primer sets, kit and the method for VEGFR1 and VEGFR2 detection of expression.
Background technology
Angiogenesis is the necessary condition of solid tumor cell growth and transfer.Vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) promotes Tumor Angiongesis and CBF by number of mechanisms Increase, it combines and activates the similar III type tyrosine kinase acceptor i.e. vascular endothelial growth factor receptor of 3 kinds of structures (Vascular Endothelial Growth Factor Receptor, VEGFR), VEGFR families include 3 kinds of hypotypes, i.e., VEGFR1 (F1t-1), VEGFR2 (KDR/Flk-1) and VEGFR3 (Flt-4).The tumor vascular generation of wherein VEGFR1 regulations is simultaneously Influence the quantity of capilary；VEGFR2 participates in the breeding of mediation Human Umbilical Vein Endothelial Cells, the formation of induced tumor blood vessel；VEGFR3 The formation of adjustable lymphangiogenesis.Therefore VEGF and VEGFR particularly VEGFR1 and VEGFR2 are that anti-angiogenesis targeting is controlled The major target for the treatment of.
The angiogenesis for suppressing tumor cell induction is the important research direction for finding new type antineoplastic medicine in recent years.Shellfish It is the 1st VEGF targeted drug Anti-X activity to cut down monoclonal antibody (Bevacizumab), and its drug mechanism is mainly logical Cross suppress tumour cell mediation angiogenesis come reach prevent tumour growth, transfer and recurrence purpose.Clinical research is Confirm, VEGFR can use the predictive factor of bevacizumab combined treatment clinical efficacy independence as colon cancer patient：I.e. The middle position progression free survival phase of the high patient of VEGFR gene expression doses is longer, and in the low patient of VEGFR gene expression doses Position progression free survival phase is shorter.
Such medicine of FDA approvals at present also includes Sutent (Sunitinib), Sorafenib (Sorafenib).Rope La Feini can suppress Raf kinases, moreover it is possible to suppress VEGFR, including VEGFR-2, VEGFR-3, platelet derived growth factor (PDGFR) etc..Sutent is a kind of small molecule TKI, is combined after energy and VEGFR tyrosine residue phosphorylations, suppresses signal and passes Lead.There are some researches show for liver cancer patient when using Sorafenib, VEGFR expression has preferably treatment curative effect compared with powerhouse.Cause This, the detection of VEGFR mRNA expressions, which receives patient targeted therapy, obvious directive significance.
At present, in the actual detection method of clinical disease mark, main immunohistochemical technique (chemical staining), stream Formula cell technology, immune radiating method etc., technology popularization is good, observation is directly perceived and relatively conventional, but it is excessively numerous experimentation to be present It is trivial as a result read the shortcomings of subjective impact is larger, it is necessary to reagent type is various, this method is limited to a certain extent clinically Further apply.In recent years, Real Time-PCR are by detection time is short, high detection sensitivity (is immunohistochemical staining More than 10 times of method), and real-time online detection, the initial content of response gene in the tissue can be carried out to PCR, experiment is saved Substantial amounts of detection time, it is thus also avoided that gradual clinically part substitution conventional inspection side the advantages that the generation of carryover contamination Method.Common methods have SYBR GreenI dye methods, double probe hybrid methods and Taqman probe techniques；Wherein SYBR GreenI methods Due to being non-saturable dye, specificity must be sentenced not as double probe hybrid methods and Taqman methods by observing solubility curve Breaking, it is specific；And two probe method hybrid method cost is costly.
In summary, the detection kit that the present invention is combined using TAQMAN probe techniques with real-time fluorescence PCR technology, Suitable for clinical expansion and industrialization.
The content of the invention
It is excessively cumbersome, it is necessary to reagent kind in order to experimentation be present when solving Immunohistochemical Method detection clinical biomarkers thing Class is various, as a result reads the larger technical problem of subjective impact, present invention offer one kind is simple to operate, detection time is short, detection Sensitiveness height and the specific good primer sets for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, reagent Box and method.
The invention provides a kind of primer sets for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, Specific primer and probe including being directed to VEGFR1 genes, VEGFR2 genes and reference gene Actin respectively, it is as follows：
VEGFR1-RF sense primers：
5'-AATCCTTCCTAAACCCAATGACTTC-3'(SEQ ID NO.1)；
VEGFR1-RR anti-sense primers：
5'-GTACGTGATGCATTGGGTGATC-3'(SEQ ID NO.2)；
VEGFR1-RP probes：
5'FAM-CTGCTCCAACCCCCGCCACC-TRAMA-3'(SEQ ID NO.3)；
VEGFR2-RF sense primers：
5'-AACCAGACAAGCGGCTACCA-3'(SEQ ID NO.4)；
VEGFR2-RR anti-sense primers：
5'-ACCGGTTTGCACTCCAATCTC-3'(SEQ ID NO.5)；
VEGFR2-RP probes：
5'FAM-ATCACTCCGATGACACAGACACCACCG-TRAMA-3'(SEQ ID NO.6)；
Actin-RF sense primers：
5'-CCGACAGGATGCAGAAGGAG-3'(SEQ ID NO.7)；
Actin-RR anti-sense primers：
5'-AGGATGGAGCCGCCGAT-3'(SEQ ID NO.8)；
Actin-RP probes：
5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'(SEQ ID NO.9)。
Present invention also offers a kind of reagent for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Box, including PCR reaction solutions, the PCR reaction solutions include detection VEGFR1 genes, VEGFR2 genes and reference gene Actin Specific primer and probe, it is as follows：
VEGFR1-RF sense primers：
5'-AATCCTTCCTAAACCCAATGACTTC-3'；
VEGFR1-RR anti-sense primers：
5'-GTACGTGATGCATTGGGTGATC-3'；
VEGFR1-RP probes：
5'FAM-CTGCTCCAACCCCCGCCACC-TRAMA-3'；
VEGFR2-RF sense primers：
5'-AACCAGACAAGCGGCTACCA-3'；
VEGFR2-RR anti-sense primers：
5'-ACCGGTTTGCACTCCAATCTC-3'；
VEGFR2-RP probes：
5'FAM-ATCACTCCGATGACACAGACACCACCG-TRAMA-3'；
Actin-RF sense primers：
5'-CCGACAGGATGCAGAAGGAG-3'；
Actin-RR anti-sense primers：
5'-AGGATGGAGCCGCCGAT-3'；
Actin-RP probes：
5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'；
The PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.
In a kind of preferred embodiment of the kit provided by the invention, in addition to positive reference substance, negative control Product and blank control product.
In a kind of preferred embodiment of the kit provided by the invention, the positive reference substance is respectively to contain The solution of VEGFR1 and VEGFR2 genes；The negative controls are the solution without VEGFR1 and VEGFR2 genes；The sky White reference substance is ultra-pure water.
Present invention also offers a kind of method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, Comprise the following steps：
Step 1：Sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, mould is used as using cDNA Plate；
Step 2：Above-mentioned kit is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions and the appropriate mould Plate mixes；
Step 3：Real-time fluorescence quantitative PCR is carried out using above-mentioned specific primer and probe, records Ct values, and respectively Standard curve is built using VEGFR1 and VEGFR2 standard genes as positive control, the bottom of sample is calculated according to the Ct values of sample Thing concentration；The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, is finally judged in sample to be detected MRNA expressions.
In a kind of preferred embodiment of methods described provided by the invention, the reaction bar of the real-time fluorescence quantitative PCR Part is：95 DEG C of pre-degeneration 5min；95 DEG C of 15sec, 60 DEG C of 30sec, fluorescence signal is caught, circulated 40 times.
Compared to prior art, express and examine provided by the present invention for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 The beneficial effect of the primer sets of survey, kit and method is：
First, the primer and TAQMAN-MGB fluorescence probe high by designing specificity, it is reconfigured to easy to use, detection knot The reliable kit of fruit, design scientific and reasonable PCR reaction systems so that the present invention have simple to operate, detection speed it is fast, The advantages that detection sensitivity is high and specific good, is particularly suitable for the popularization and application in clinical examination works；
2nd, it is reference gene by increasing Actin genes in design of primers and kit, is had based on the reference gene The advantages of mRNA expression is stable, it is possible to prevente effectively from the generation of false negative, and preferably destination gene expression amount can also be carried out Correction, so as to improve the sensitivity of the inventive method and repeatability so that testing result is more accurately and reliably；
3rd, the above-mentioned advantage having based on the present invention, it can cause the present invention that clinically there is good economy and society Can benefit, the particularly personalized medicine to clinical tumor targeted drug (such as Cetuximab, Herceptin, Lapatinib) Have and know meaning well, important reference frame can be provided for clinical precisely medication, reduce the unreasonable feelings used of medicine Shape, the medical treatment cost of the whole society is reduced, save society doctor and defend resource.
Brief description of the drawings
The PCR amplification curve diagrams of Actin mRNA when Fig. 1 is clinical same pattern detection；
The PCR amplification curve diagrams of VEGFR 1mRNA when Fig. 2 is clinical same pattern detection；
The PCR amplification curve diagrams of Actin mRNA when Fig. 3 is clinical another pattern detection；
The PCR amplification curve diagrams of VEGFR 2mRNA when Fig. 4 is clinical another pattern detection.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and It is not used in the restriction present invention.
Embodiment 1：The preparation of kit
First, the design and synthesis of primer and probe
For VEGFR1 genes, VEGFR2 genes and reference gene Actin, (sequence is referring to disclosed in ncbi database respectively Mankind's whole genome sequence), use Primer Premier 3.0 and Methyl Primer Express v1.0 softwares, difference Specific primer and probe are designed, probe is marked using 5'-FAM and 3'-TRAMAMGB.
Specific primer and probe sequence, it is as shown in the table：
2nd, reference substance selects
Positive reference substance is respectively the solution of the gene containing VEGFR1 and VEGFR2；Negative controls be without VEGFR1 and The solution of VEGFR2 genes；Blank control product are ultra-pure water.
3rd, PCR reaction solutions form
Including the PCR reaction solutions containing above-mentioned specific primer and probe, the PCR reaction solutions include detection VEGFR1 bases The specific primer and probe of cause, VEGFR2 genes and reference gene Actin；Also include PCR buffer, dNTP, HS Taq enzymes And ultra-pure water.
Wherein, it is (precious biological (big to be purchased from the precious biology in Dalian for PCR buffer, dNTP, HS Taq enzyme and ultra-pure water Even) Engineering Co., Ltd)；The ultra-pure water is that (Nuclease-Free Water) uses DEPC (Diethyl Pyrocarbonate, pyrocarbonic acid diethyl ester) the treated and ultra-pure water through autoclave sterilization；HS Taq enzymes (TaKaRa Taq Hot Start Version) it is to be directed to general T aq archaeal dna polymerase high sensitivities, the situation of non-specific band is also easy to produce, specially The thermophilic archaeal dna polymerase product of high specific that door is developed.
4th, PCR reaction systems configure
Embodiment 2：Clinical tumor targeted drug (such as Cetuximab, Herceptin, drawing are carried out using mentioned reagent box Pa replaces Buddhist nun) the horizontal detection of personalized medicine related gene expression method
First, biological specimen
Biomaterial of the present invention is all from Guangdong and attains medical test institute greatly.For during the 1-12 months in 2016 wide Attain 100 remaining tissue samples that medical test is detected greatly in east.
2nd, sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, template is used as using cDNA：
Sample mRNA extracts kits are QIAGEN miRNeasy FFPE Kit kit (paraffin-embedded tissue samples RNA is extracted) (Kai Jie Bioisystech Co., Ltd (Shanghai)) and QIAGEN RNeasy Mini Kit kit (flesh tissue samples This RNA is extracted) (Kai Jie Bioisystech Co., Ltd (Shanghai))；
Sample mRNA Reverse Transcriptase kits are that (treasured is raw for Takara-PrimeScriptTM RT reagent Kit kits Thing engineering (Dalian) Co., Ltd).
1) take and receive qualified clinical detection sample (the normal structure sample and tumor tissues sample each one of same patient Part)；
2) reagent used required for extraction mRNA is taken out from mRNA extracts kits, normal structure sample is extracted and swells The mRNA of tumor tissue sample；
3) the mRNA samples of extraction need to carry out reverse transcription operation in 24 hours, otherwise need to be placed in -80 DEG C of preservations；
4) reagent used required for reverse transcription mRNA is taken out from mRNA Reverse Transcriptase kits, by extraction in step 3) MRNA reverse transcriptions are cDNA, and are used as template using cDNA.
The 3rd, the kit of the present invention is provided, take out appropriate PCR reaction solutions, by the PCR reaction solutions and the appropriate template Mix；
1) fluorescence quantitative PCR reaction solution, Taq enzyme, primer, probe are taken out from kit, room temperature is melted and vibrates mixing Afterwards, 2000rpm centrifuges 10sec；
It is determined that detection reaction tube number is n (standard items+negative control of the pipe various concentrations gradient of n=sample numbers+4) by following Single detection reaction system prepares the dosage that meter calculates reaction system (40ul) each reagent, adds a proper volume centrifuge tube, fills Divide and mix, be distributed into after 2000rpm centrifugations 10sec in each PCR reaction tubes, 38 μ l/ pipes；Pay attention to avoiding producing bubble, cover Specimen Treatment Chamber is transferred to after lid.
Single detection reaction system is with tabulation：
2) sample rna reverse transcription product is taken out, puts thaw at RT, 2000rpm centrifugations 10sec；Divide in PCR reaction tubes Not Jia Ru in normal sample RNA reverse transcription products, lesion sample rna reverse transcription product and kit various concentrations gradient standard The μ l of product 2；2ul sterilizing ultra-pure waters are added in negative control pipe；Sample should be directly added into reaction solution, avoid sample adhesion in pipe On wall, low-speed centrifugal 10sec after lid fully mixes is covered tightly, is transferred to amplification region.
3) PCR reaction tubes are sequenced and is put into ABI7500 quantitative real time PCR Instruments (U.S. AppliedBiosystems public affairs Department)；PCR response parameters are set；
Cycling condition is set：
Reaction system is 40 μ l.Instrument sense channel selects：Fluorescence signal collection：Fam fluoresceins；Collect temperature：60℃. (should strictly ensure that the normal sample of same patient is consistent with the amplification condition of lesion sample)
4th, PCR result judgements：
The interpretation of result software of quantitative real time PCR Instrument is opened, imports the experimental data of preservation；Using spot fitting method, enter Row interpretation of result, build the standard curve of standard items；Carry out the determination of interpretation of result base line：Take what 3-10 or 3-15 were circulated Fluorescence signal.Threshold value can adjust according to instrument noise tolerance limit.Threshold value setting principle is negative right just above normal with threshold line It is defined according to the peak of product amplification curve；Missing inspection strong positive sample is avoided, baseline should be set to 3-10 circulation fluorescence letter first Number observation analysis Ct values, if without Ct values ＜ 16.0 sample, baseline is changed and is set to the fluorescence signal of 3-15 circulation and enters Row interpretation of result.
Quality control standard：The testing result of negative control should be negative；The Ct values of blank control should be equal to 40.0；Otherwise, this It is invalid that secondary experiment is considered as.The coefficient correlation of standard curve is more than 0.99；Otherwise, this time experiment is considered as invalid.
As a result judge：
The standard curve built according to standard items, and calculate according to the Ct values of sample the concentration of substrate of sample.
The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, it can be determined that patient's pathological tissues MRNA expressions in sample.
Comparative result VEGFR1, VEGFR2mRNA expression
Normal sample concentration is less than lesion concentration of specimens Pathological tissues mRNA is high level expression
Normal sample concentration is higher than lesion concentration of specimens Pathological tissues mRNA is low expression level
Clinical meaning：
Clinical diagnosis finds that VEGFR1, VEGFR2 mrna expression receive cancer patient the treatment of targeted therapy Effect has obvious directive significance, has preferably treatment curative effect when the patient of height expression is using Sorafenib, Sutent, low The patient of expression uses curative effect unobvious when Sorafenib, Sutent.
Based on the clinical sample mRNA expression testing results of the present invention, referring to Fig. 1-Fig. 3, wherein Fig. 1-2 is clinic The PCR amplification curve diagrams of Actin mRNA during same pattern detection and VEGFR1mRNA PCR amplification curve diagrams；Fig. 3-4 is Actin mRNA PCR amplification curve diagrams and VEGFR 2mRNA PCR amplification curve diagrams during clinical another pattern detection.
Primer sets, reagent provided by the present invention for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression The beneficial effect of box and method is：
First, the primer and TAQMAN-MGB fluorescence probe high by designing specificity, it is reconfigured to easy to use, detection knot The reliable kit of fruit, design scientific and reasonable PCR reaction systems so that the present invention have simple to operate, detection speed it is fast, The advantages that detection sensitivity is high and specific good, is particularly suitable for the popularization and application in clinical examination works；
2nd, it is reference gene by increasing Actin genes in design of primers and kit, is had based on the reference gene The advantages of mRNA expression is stable, it is possible to prevente effectively from the generation of false negative, and preferably destination gene expression amount can also be carried out Correction, so as to improve the sensitivity of the inventive method and repeatability so that testing result is more accurately and reliably；
3rd, the above-mentioned advantage having based on the present invention, it can cause the present invention that clinically there is good economy and society Can benefit, the particularly personalized medicine to clinical tumor targeted drug (such as Cetuximab, Herceptin, Lapatinib) Have and know meaning well, important reference frame can be provided for clinical precisely medication, reduce the unreasonable feelings used of medicine Shape, the medical treatment cost of the whole society is reduced, save society doctor and defend resource.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap Include in the scope of patent protection of the present invention.
Sequence table
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Claims (6)

  1. A kind of 1. primer sets for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, it is characterised in that bag The specific primer and probe for being directed to VEGFR1 genes, VEGFR2 genes and reference gene Actin respectively are included, it is as follows：
    VEGFR1-RF sense primers：
    5'-AATCCTTCCTAAACCCAATGACTTC-3'；
    VEGFR1-RR anti-sense primers：
    5'-GTACGTGATGCATTGGGTGATC-3'；
    VEGFR1-RP probes：
    5'FAM-CTGCTCCAACCCCCGCCACC-TRAMA-3'；
    VEGFR2-RF sense primers：
    5'-AACCAGACAAGCGGCTACCA-3'；
    VEGFR2-RR anti-sense primers：
    5'-ACCGGTTTGCACTCCAATCTC-3'；
    VEGFR2-RP probes：
    5'FAM-ATCACTCCGATGACACAGACACCACCG-TRAMA-3'；
    Actin-RF sense primers：
    5'-CCGACAGGATGCAGAAGGAG-3'；
    Actin-RR anti-sense primers：
    5'-AGGATGGAGCCGCCGAT-3'；
    Actin-RP probes：
    5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'。
  2. A kind of 2. kit for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, it is characterised in that bag PCR reaction solutions are included, the PCR reaction solutions include detection VEGFR1 genes, VEGFR2 genes and reference gene Actin specificity Primer and probe, it is as follows：
    VEGFR1-RF sense primers：
    5'-AATCCTTCCTAAACCCAATGACTTC-3'；
    VEGFR1-RR anti-sense primers：
    5'-GTACGTGATGCATTGGGTGATC-3'；
    VEGFR1-RP probes：
    5'FAM-CTGCTCCAACCCCCGCCACC-TRAMA-3'；
    VEGFR2-RF sense primers：
    5'-AACCAGACAAGCGGCTACCA-3'；
    VEGFR2-RR anti-sense primers：
    5'-ACCGGTTTGCACTCCAATCTC-3'；
    VEGFR2-RP probes：
    5'FAM-ATCACTCCGATGACACAGACACCACCG-TRAMA-3'；
    Actin-RF sense primers：
    5'-CCGACAGGATGCAGAAGGAG-3'；
    Actin-RR anti-sense primers：
    5'-AGGATGGAGCCGCCGAT-3'；
    Actin-RP probes：
    5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'；
    The PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.
  3. 3. the reagent according to claim 2 for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Box, it is characterised in that also including positive reference substance, negative controls and blank control product.
  4. 4. the reagent according to claim 3 for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Box, it is characterised in that the positive reference substance is respectively the solution of the gene containing VEGFR1 and VEGFR2；The negative controls are Solution without VEGFR1 and VEGFR2 genes；The blank control product are ultra-pure water.
  5. A kind of 5. method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, it is characterised in that including Following steps：
    Step 1：Sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, template is used as using cDNA；
    Step 2：Kit as claimed in claim 4 is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions with fitting The template is measured to mix；
    Step 3：Real-time fluorescence quantitative PCR is carried out using the specific primer described in claim 1 and probe, records Ct values, and Standard curve is built using VEGFR1 and VEGFR2 standard genes as positive control respectively, sample is calculated according to the Ct values of sample Concentration of substrate；The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, finally judges test sample to be checked MRNA expressions in this.
  6. 6. the method according to claim 5 for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression, Characterized in that, the reaction condition of the real-time fluorescence quantitative PCR is：95 DEG C of pre-degeneration 5min；95 DEG C of 15sec, 60 DEG C 30sec, fluorescence signal is caught, circulated 40 times.
CN201711220736.XA 2017-11-29 2017-11-29 Primer sets, kit and method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression Pending CN107858431A (en)

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CN108949981A (en) * 2018-07-17 2018-12-07 济南艾迪康医学检验中心有限公司 Utilize the kit of real-time fluorescence quantitative PCR detection VEGFR2 gene relative expression quantity

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Application publication date: 20180330