CN103627814B - Reagent for detecting Notch signal path as well as PCR (Polymerase Chain Reaction) detecting method and application thereof - Google Patents
Reagent for detecting Notch signal path as well as PCR (Polymerase Chain Reaction) detecting method and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
The invention provides a reagent for detecting a Notch signal path as well as a PCR (Polymerase Chain Reaction) detecting method and application thereof. The detecting reagent comprises PCR reaction primers for detecting an ADAM10 gene, an ADAM17 gene, an AES gene, a CBL gene, a CCND1gene, a CD44 gene, and the like, and primers of corresponding internal-reference regulatory genes GAPDH (Reduced Glyceraldehyde-Phosphate Dehydrogenase), ACTB (Beta-Actin) and B2M (Beta-2-Microglobulin). The invention provides the reagent for detecting core molecule changes of the NOTCH signal channel; by virtue of the detecting reagent, genes associated with the NOTCH channel can be quickly detected on transcriptional level by virtue of real-time fluorescent quantitative PCR; on this basis, core molecules and a mechanism of action of a tumor NOTCH signal channel are analyzed and researched, so that an NOTCH regulatory channel of tumor-associated medicaments is quickly and accurately found, and therefore, a powerful tool is provided for screening anti-tumor medicaments, discussing the mechanism of a novel targeted medicament, and the like.
Description
Technical field
The invention belongs to biology field, particularly relate to a kind of detect Notch signal path reagent, PCR detection method and application thereof.
Background technology
Notch signal is high conservative in a kind of genetic evolution, a kind of signal path of communication role between reflection flanking cell, it not only plays an important role in cell normal development, differentiation and proliferation and apoptosis, and has dependency with the generation of kinds of tumors and development.
Notch signal path is made up of associated proteins and target gene in the acceptor of cross-film, part and core, Notch acceptor comprises Notch extracellular domain and cross-film district/Notch intracellular domain, is the single transmembrane albumen with transcriptional control dual-use function in cell surface receptor and core.Current discovery has 4 kinds of Notch acceptors, i.e. Notch-l, Notch-2, Notch-3 and Notch-4 at mammalian, and they are usually mutually triggered by the Notch part of flanking cell and activate.Notch part is also the single transmembrane albumen being expressed in cell surface, is named as Delta and Serrate, in nematode, is named as Lag-2 in fruit bat, and therefore Notch part has another name called DSL(Delta/Serrate/Lag-2) albumen.DSL high conservative, Delta or Delta sample (Delta-like is called as with the part of Delta very high homology in Mammals, DLL), with Serrate very high homology be called as Serrate or Jagged, namely 5 kinds of parts are respectively Delta-1, Delta-3, Delta-4, Jagged-1 and Jagged-2, with the combination and reactivation process of flanking cell Notch acceptor in there is key effect.To be triggered by corresponding part when Notch acceptor and be combined with its DSL structural domain, after 2 proteolytic cleavages that gamma-secretase etc. mediates, the NICD of the activated form of Notch acceptor-have nuclear localization signal is released in tenuigenin, and transposition is transferred to nucleus, by being combined with transcription factor, play the regulating and controlling effect to target gene.
The generation of tumour and development are the coefficient results of many factors.Increasing evidence display in recent years, Notch signal path is except outside the Pass having with the generation and development of multiple solid tumor, the Notch signal path activated in enteron aisle can form intestinal tumor by promoting tumor stem cell propagation, show that Notch signal also plays very crucial effect in the formation of digestive system tumor, and different roles is play in the dissimilar of same tumour or different stages of development, both can show as tumorigenicity, can show as again press down carcinous.
Although Notch signal plays an important role in the process of origin of life, also show in minority tumour press down carcinous, but generation and the development of the Notch signal of exception and tumour have contacting of countless ties, to Notch signal and relative disease, molecular mechanism particularly and between tumour, needs to be illustrated further.The effect and the exploitation that play Notch signal path cancer suppressor gene to greatest extent have broad application prospects with the new drug that Notch signal path is target spot, also open up new approach by for oncotherapy.
Summary of the invention
The present invention is directed to the regulation and control how determining NOTCH core signal molecule in prior art, how to explain the difficulty of the change of core element in tumour cell, provide a kind of reagent and the PCR detection method that detect Notch signal path, the present invention will relate to apoptotic signaling molecule and concentrate on a flat board, by doing a real-time fluorescence quantitative PCR reaction, the survival condition of reacting cells, inquire in breast cancer cell line, cause NOTCH signal path to change possible target molecule and target approach, for the adjustment of research core NOTCH signal protein provides the most direct evidence.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
Detect a reagent for Notch signal path, it comprises following primer:
(1) PCR detecting ADAM10 gene reacts primer, and its primer sequence is as follows:
5'-ATGGGAGGTCAGTATGGGAAT-3'；
5'-ACTGCTCTTTTGGCACGC-3'；
(2) PCR detecting ADAM17 gene reacts primer, and its primer sequence is as follows:
5'-ATGTTTCACGTTTGCAGTCTC-3'；
5'-AGAAGCGATGATCTGCTACC-3'；
(3) detect AES gene PCR react primer, its primer sequence is as follows:
5'-ACCCCAGCAACTCAAATTCA-3'；
5'-AAGCCGTAGGACATCTCGTA-3'；
(4) detect CBL gene PCR react primer, its primer sequence is as follows:
5'-TGGTGCGGTTGTGTCAGA-3'；
5'-GGTAGGTATCTGGTAGCAGG-3'；
(5) detect CCND1 gene PCR react primer, its primer sequence is as follows:
5'-GCTGCGAAGTGGAAACCAT-3'；
5'-CCTCCTTCTGCACACATTTGA-3'；
(6) detect CD44 gene PCR react primer, its primer sequence is as follows:
5'-CCCATCCCAGACGAAGACA-3'；
5'-GCCATTTGTGTTGTTGTGTGA-3'；
(7) PCR detecting CDKN1A gene reacts primer, and its primer sequence is as follows:
5'-TGTCCGTCAGAACCCATG-3'；
5'-AAAGTCGAAGTTCCATCGCT-3'；
(8) PCR detecting CFLAR gene reacts primer, and its primer sequence is as follows:
5'-AGAGTGAGGCGATTTGACCT-3'；
5'-GTCCGAAACAAGGTGAGGGT-3'；
(9) PCR detecting CHUK gene reacts primer, and its primer sequence is as follows:
5'-ATGAAGAAGTTGAACCATGCC-3'；
5'-CCTCCAGAACAGTATTCCATTG-3'；
(10) PCR detecting CTNNB1 gene reacts primer, and its primer sequence is as follows:
5'-ATGTCCAGCGTTTGGCTGA-3'；
5'-TGGTCCTCGTCATTTAGCAGT-3'；
(11) PCR detecting DLL1 gene reacts primer, and its primer sequence is as follows:
5'-TGTGACGAACACTACTACGG-3'；
5'-AGCCAGGGTTGCACACTT-3'。
(12) PCR detecting DLL3 gene reacts primer, and its primer sequence is as follows:
5'-GCCAGAGCTTTTCCACTGATT-3'；
5'-GCGTCAGGCCTAGAAACAAAA-3'；
(13) PCR detecting DLL4 gene reacts primer, and its primer sequence is as follows:
5'-ACCTTCTCGCTCATCATCGA-3'；
5'-CCTGGATGGCGATCTTGC-3'；
(14) PCR detecting DTX1 gene reacts primer, and its primer sequence is as follows:
5'-CAGTCCATGCACCAGTTTC-3'；
5'-TCCATATCGTAGGCCGTCCA-3'；
(15) PCR detecting ERBB2 gene reacts primer, and its primer sequence is as follows:
5'-GCTCATCGCTCACAACCAAG-3'；
5'-ACAGGGGTGGTATTGTTCAG-3'；
(16) PCR detecting FOSL1 gene reacts primer, and its primer sequence is as follows:
5'-CAGGCGGAGACTGACAAACT-3'；
5'-TCCTTCCGGGATTTTGCAGA-3'；
(17) PCR detecting FZD1 gene reacts primer, and its primer sequence is as follows:
5'-ATCTTCTTGTCCGGCTGTTAC-3'；
5'-GTCCTCGGCGAACTTGTCAT-3'；
(18) PCR detecting FZD2 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCATCCTATCTCAGCTAC-3'；
5'-CTGCATGTCTACCAAGTACGT-3'；
(19) PCR detecting FZD3 gene reacts primer, and its primer sequence is as follows:
5'-GGATTGTTCTCGGGATTTCC-3'；
5'-AGTGTGACACGTCCATATTCC-3'；
(20) PCR detecting FZD4 gene reacts primer, and its primer sequence is as follows:
5'-GTCTTTCAGTCAAGAGACGCT-3'；
5'-GTTGTGGTCGTTCTGTGGT-3'；
(21) PCR detecting FZD6 gene reacts primer, and its primer sequence is as follows:
5'-GTGCAACTCTGTTCACATTCC-3'；
5'-GCTATCGCCTAGCAAAAATCC-3'；
(22) PCR detecting FZD7 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCAACGGCCTGATGT-3'；
5'-AGGTGAGAACGGTAAAGAGC-3'；
(23) PCR detecting GBP2 gene reacts primer, and its primer sequence is as follows:
5'-TTTCCAGCATTTGTGTGGAC-3'；
5'-GGGAAGAACTTTCGGATGCA-3'；
(24) PCR detecting Gli1 gene reacts primer, and its primer sequence is as follows:
5'-TTCCTACCAGAGTCCCAAG-3'；
5'-CCCTATGTGAAGCCCTATT-3'；
(25) PCR detecting GSK3B gene reacts primer, and its primer sequence is as follows:
5'-GGCAGCATGAAAGTTAGCAG-3'；
5'-GGCGACCAGTTCTCCTGAAT-3'；
(26) PCR detecting HES1 gene reacts primer, and its primer sequence is as follows:
5'-ACACGACACCGGATAAACCA-3'；
5'-GCCGCGAGCTATCTTTCTTC-3'；
(27) PCR detecting HEY1 gene reacts primer, and its primer sequence is as follows:
5'-ATCTGCTAAGCTAGAAAAAGCC-3'；
5'-CGTCAAAGTAACCTTTCCCTCC-3'；
(28) PCR detecting HEYL gene reacts primer, and its primer sequence is as follows:
5'-GGCTGCTTACGTGGCTGT-3'；
5'-GACCCAGGAGTGGTAGAGCA-3'；
(29) PCR detecting HOXB4 gene reacts primer, and its primer sequence is as follows:
5'-CGTGAGCACGGTAAACCC-3'；
5'-CGAGCGGATCTTGGTGTT-3'；
(30) PCR detecting HR gene reacts primer, and its primer sequence is as follows:
5'-CCCAGAGCATCCATGTGACT-3'；
5'-TGCCCACCAGAGTTTAAGC-3'；
(31) PCR detecting IFNG gene reacts primer, and its primer sequence is as follows:
5'-TCGGTAACTGACTTGAATGTCC-3'；
5'-TCGCTTCCCTGTTTTAGCTG-3'；
(32) PCR detecting IL17B gene reacts primer, and its primer sequence is as follows:
5'-GAGCCCCAAAAGCAAGAGGA-3'；
5'-TGCGGGCATACGGTTTCAT-3'；
(33) PCR detecting IL2RA gene reacts primer, and its primer sequence is as follows:
5'-GTGGGGACTGCTCACGTT-3'；
5'-GGATCTCTGGCGGGTCAT-3'；
(34) PCR detecting ITGA1 gene reacts primer, and its primer sequence is as follows:
5'-CTGGACATAGTCATAGTGCTGG-3'；
5'-ACCTGTGTCTGTTTAGGACC-3'；
(35) PCR detecting ITGA2 gene reacts primer, and its primer sequence is as follows:
5'-GGGAATCAGTATTACACAACGG-3'；
5'-CCACAACATCTATGAGGGAAGG-3'；
(36) PCR detecting ITGA3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGCTTGGAGTGACTGT-3'；
5'-TCATTGCCTCGCACGTAG-3'；
(37) PCR detecting ITGA4 gene reacts primer, and its primer sequence is as follows:
5'-GGTAGCCCTAATGGAGAACC-3'；
5'-TATGCCCACAAGTCACGATG-3'；
(38) PCR detecting ITGAV gene reacts primer, and its primer sequence is as follows:
5'-CCCCATTGTACCATTGGAGAA-3'；
5'-TGGAGCATACTCAACAGTCTTT-3'；
(39) PCR detecting ITGB1 gene reacts primer, and its primer sequence is as follows:
5'-CAAGAGAGCTGAAGACTATCCC-3'；
5'-TGAAGTCCGAAGTAATCCTCC-3'；
(40) PCR detecting ITGB3 gene reacts primer, and its primer sequence is as follows:
5'-GTGACCTGAAGGAGAATCTG-3'；
5'-CCGGAGTGCAATCCTCTG-3'；
(41) PCR detecting ITGB5 gene reacts primer, and its primer sequence is as follows:
5'-GGAAGTTCGGAAACAGAGGG-3'；
5'-CTTTCGCCAGCCAATCTTCT-3'；
(42) PCR detecting JAG1 gene reacts primer, and its primer sequence is as follows:
5'-GGGGCAACACCTTCAACCT-3'；
5'-CCAGGCGAAACTGAAAGG-3'；
(43) PCR detecting JAG2 gene reacts primer, and its primer sequence is as follows:
5'-TGGGCGGCAACTCCTTCT-3'；
5'-GCCTCCACGATGAGGGTAA-3'；
(44) PCR detecting KAT2B gene reacts primer, and its primer sequence is as follows:
5'-GAAAAACCCTAACCCCTCAC-3'；
5'-CCTTGTGGACACAGGTAAAGAG-3'；
(45) PCR detecting KRT1 gene reacts primer, and its primer sequence is as follows:
5'-GGTGCTTATATGACCAAGGTG-3'；
5'-ATGCTGTCCAGGTCGAGAC-3'；
(46) PCR detecting LFNG gene reacts primer, and its primer sequence is as follows:
5'-GTCAGCGAGAACAAGGTG-3'；
5'-GATCCGCTCAGCCGTATTCA-3'；
(47) PCR detecting LMO2 gene reacts primer, and its primer sequence is as follows:
5'-GACAAGCGGATTCGTGCCTA-3'；
5'-AGTTGATGAGGAGGTATCTGTC-3'；
(48) PCR detecting MAP2K7 gene reacts primer, and its primer sequence is as follows:
5'-TGCTTTGGGACGTTCATCAC-3'；
5'-CCACTGTCATCTTGCCCAGA-3'；
(49) PCR detecting MFNG gene reacts primer, and its primer sequence is as follows:
5'-TGCTGAGTTCGACACCTTCT-3'；
5'-GCCCTTGGGTTCACATAGTT-3'；
(50) PCR detecting MMP7 gene reacts primer, and its primer sequence is as follows:
5'-GAGTGAGCTACAGTGGGAAC-3'；
5'-CTATGACGCGGGAGTTTAACA-3'；
(51) PCR detecting MYCL1 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCCCAAACCTGTCA-3'；
5'-GGCCTTGCTTAGGATCACTACT-3'；
(52) PCR detecting NCOR2 gene reacts primer, and its primer sequence is as follows:
5'-TGCAGATCATCTACGACGAG-3'；
5'-TCCGCATCGCCTGGTTTATT-3'；
(53) PCR detecting NESTIN gene reacts primer, and its primer sequence is as follows:
5'-AGCCCTGACCACTCCAGTTTA-3'；
5'-CCCTCTATGGCTGTTTCTTTCTC-3'；
(54) PCR detecting NEURL gene reacts primer, and its primer sequence is as follows:
5'-GTGTCTTCCACCGCATCAA-3'；
5'-ACCAGCTCGCTATCAAGCA-3'；
(55) PCR detecting NFKB1 gene reacts primer, and its primer sequence is as follows:
5'-GAAGCACGAATGACAGAGG-3'；
5'-GCTTGGCGGATTAGCTCTTT-3'；
(56) PCR detecting NFKB2 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAGAGTTGCTACAACCC-3'；
5'-CTGTTCCACGATCACCAGGT-3'；
(57) PCR detecting Notch1 gene reacts primer, and its primer sequence is as follows:
5'-ATCGACGATTGTCCAGGAAAC-3'；
5'-TACTGACCTGTCCACTCTGGC-3'；
(58) PCR detecting NOTCH2 gene reacts primer, and its primer sequence is as follows:
5'-AAGCAGAGTCCCAGTGCCT-3'；
5'-CAGGGGGCACTGACAGTAA-3'；
(59) PCR detecting NOTCH2NL gene reacts primer, and its primer sequence is as follows:
5'-TGTGACAGCCTGTATGTGC-3'；
5'-TGAGCAGCTTAGGAAAGATTG-3'；
(60) PCR detecting NOTCH3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGACGAGTGCTCTATC-3'；
5'-AATGTCCACCTCGCAATAG-3'；
(61) PCR detecting NOTCH4 gene reacts primer, and its primer sequence is as follows:
5'-CTAGGGGCTCTTCTCGTCC-3'；
5'-CAACTTCTGCCTTTGGCTT-3'；
(62) PCR detecting NR4A2 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCTTGCTTGTACCAAA-3'；
5'-GGCTTGTAGTAAACCGACCC-3'；
(63) PCR detecting NUMB gene reacts primer, and its primer sequence is as follows:
5'-TCAGCAGATGGACTCAGAGT-3'；
5'-AGGCTCTATCAAAGTTCCTGTC-3'；
(64) PCR detecting PAX5 gene reacts primer, and its primer sequence is as follows:
5'-ACTTGCTCATCAAGGTGTCA-3'；
5'-TCCTCCAATTACCCCAGGCT-3'；
(65) PCR detecting PDPK1 gene reacts primer, and its primer sequence is as follows:
5'-ACGGAGAAGTCCGCCTGTA-3'；
5'-CTCGTTTCCAGCTCGGAAT-3'；
(66) PCR detecting POFUT1 gene reacts primer, and its primer sequence is as follows:
5'-AACCAGGCCGATCACTTCTT-3'；
5'-GTTGGTGAAAGGAGGCTTGT-3'；
(67) PCR detecting PPARG gene reacts primer, and its primer sequence is as follows:
5'-GGGATCAGCTCCGTGGATC-3'；
5'-TGCACTTTGGTACTCTTGAAGT-3'；
(68) PCR detecting PSEN1 gene reacts primer, and its primer sequence is as follows:
5'-GACGACCCCAGGGTAACT-3'；
5'-ACTGACTTAATGGTAGCCACG-3'；
(69) PCR detecting PSEN2 gene reacts primer, and its primer sequence is as follows:
5'-ACCGCTATGTCTGTAGTG-3'；
5'-CGCTCCGTATTTGAGGGTCA-3'；
(70) PCR detecting PSENEN gene reacts primer, and its primer sequence is as follows:
5'-CTGGAGCGAGTGTCCAATGA-3'；
5'-GCGCCAGACATAGCCTTTGA-3'；
(71) PCR detecting RFNG gene reacts primer, and its primer sequence is as follows:
5'-CGCCAGCAGACGTTTATCTT-3'；
5'-CCGAGCAGTTGGTGTTGAT-3'；
(72) PCR detecting RUNX1 gene reacts primer, and its primer sequence is as follows:
5'-CTGCCCATCGCTTTCAAGG-3'；
5'-GCCGAGTAGTTTTCATCATTGC-3'；
(73) PCR detecting SEL1L gene reacts primer, and its primer sequence is as follows:
5'-AAACCAGCTTTGACCGCCA-3'；
5'-GTCATAGGTTGTAGCACACCA-3'；
(74) PCR detecting SH2D1A gene reacts primer, and its primer sequence is as follows:
5'-GACAGCACCTGGGGTACATA-3'；
5'-TGCAGAGGTATTACAATGCCTT-3'；
(75) PCR detecting SHH gene reacts primer, and its primer sequence is as follows:
5'-CTCGCTGCTGGTATGCTC-3'；
5'-ATCGCTCGGAGTTTCTGGAG-3'；
(76) PCR detecting Smo gene reacts primer, and its primer sequence is as follows:
5'-GTTCTCCATCAAGAGCAACCA-3'；
5'-CGATTCTTGATCTCACAGTCAG-3'；
(77) PCR detecting SNW1 gene reacts primer, and its primer sequence is as follows:
5'-GGATACCGGAAAGGCTGGAT-3'；
5'-TGACTGTCCTTGTCGAGCAA-3'；
(78) PCR detecting STAT6 gene reacts primer, and its primer sequence is as follows:
5'-GTTCCGCCACTTGCCAAT-3'；
5'-GTTCCGCCACTTGCCAAT-3'；
(79) PCR detecting SUFU gene reacts primer, and its primer sequence is as follows:
5'-CCAGGTTACCGCTATCGTCA-3'；
5'-CGAAGCTGATGTAGTGCCAG-3'；
(80) PCR detecting TEAD1 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAAAGGATGAGTGACTCTG-3'；
5'-TCCCACATGGTGGATAGATAG-3'；
(81) PCR detecting TLE1 gene reacts primer, and its primer sequence is as follows:
5'-GAGTCCCTGGACCGGATTAA-3'；
5'-AATACATCACATAGTGCCTCTG-3'；
(82) PCR detecting WISP1 gene reacts primer, and its primer sequence is as follows:
5'-TGCTGTAAGATGTGCGCTCA-3'；
5'-ACACTCCTATTGCGTACCTC-3'；
(83) PCR detecting WNT11 gene reacts primer, and its primer sequence is as follows:
5'-GGAGTCGGCCTTCGTGTAT-3'；
5'-GCTGAGGTTGTCCGCACA-3'；
(84) PCR detecting ZIC2 gene reacts primer, and its primer sequence is as follows:
5'-CACCTCCGATAAGCCCTATC-3'；
5'-GGCGTGGACGACTCATAG-3'。
The PCR of described reagent also containing internal reference regulatory gene GAPDH, ACTB, B2M reacts primer.
The PCR detecting GAPDH gene reacts primer, and its primer sequence is as follows:
5'-ACTGCCACCCAGAAGAC-3'；
5'-GCTCAGTGTAGCCCAGGA-3'；
The PCR detecting ACTB gene reacts primer, and its primer sequence is as follows:
5'-CATGTACGTTGCTATCCAGG-3'；
5'-CTCCTTAATGTCACGCACGA-3'；
The PCR detecting B2M gene reacts primer, and its primer sequence is as follows:
5'-GAGGCTATCCAGCGTACTCC-3'；
5'-CGGCAGGCATACTCATCTTT-3'。
Present invention also offers the PCR detection method detecting cell NOTCH signal path by described detection reagent, described PCR detection method is real time fluorescence quantifying PCR method.
Further improvement to technique scheme: the reaction system of described real-time fluorescence quantitative PCR is: Taq polymeric enzyme reaction mixed solution 10 μ l, upstream primer 0.2 μm of ol, downstream primer 0.2 μm of ol, cDNA template 50ng, sterile purified water supplies 20 μ l.
Further improvement to technique scheme: described Taq polysaccharase is TaKaRa company
premix Ex Taq polysaccharase.
Further improvement to technique scheme: the response procedures of described real-time fluorescence quantitative PCR is: 95 DEG C of denaturations, 30 seconds; 95 DEG C of sex change, 5 seconds, 60 DEG C, 20 seconds, carry out 40 circulations.
Present invention also offers the application of described reagent in the detection reagent of preparation detection cancer cells, described cancer cells is lung carcinoma cell, breast cancer cell, lung carcinoma cell and liver cancer cell.
Compared with prior art, advantage of the present invention and positively effect are: the invention provides the reagent detecting the change of NOTCH signal path core element, the gene relevant to NOTCH can be gone out by real-time fluorescence quantitative PCR in transcriptional level rapid detection by means of described detection reagent, to analyze and research on this basis the core element of tumour NOTCH signal path and the mechanism of action, thus find the NOTCH conditioning signal path of tumour related drugs fast and accurately, for the Mechanism Discussion etc. of screening anticancer medicine, new targeted drug provides strong instrument.
Present invention also offers the PCR method of detection, described experimental system can carry out on any real-time fluorescence quantitative PCR instrument, and its PCR reaction kit is the LightCycler480 of Roche Holding Ag simultaneously.Above-mentioned primer is placed in 96 orifice plates, carries out real-time fluorescence quantitative PCR detection.Can by the change of primary first-order equation acquisition about NOTCH signal path core element.Experimental implementation is easy, low cost, and result is reproducible, is conducive to reducing the unnecessary detection in downstream, is a kind of important means of the research tumour related drugs mechanism of action.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, the other features and advantages of the invention will become clearly.
Accompanying drawing explanation
Fig. 1 indicates the present invention and utilizes NOTCH signal path to filter out the gene of breast cancer cell line HS578T and MCF-10 HS578BST differential expression, wherein 1:HS578T cell; 2:HS578BST cell.
Fig. 2 real-time fluorescence quantitative PCR detects the expression of NOTCH signal path gene.
Fig. 3 solubility curve analyzes the expression of NOTCH signal path gene.
The difference of NOTCH signal path genetic expression in Fig. 4 mammary cancer/normal breast cell.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
Embodiment 1
The present invention is illustrated by breast cancer cell, but the present invention is not limited to mammary cancer, and detection reagent of the present invention can be applied to common cancers such as comprising mammary cancer, lung cancer, liver cancer.
The present invention, by cultivation two kinds of clone: MCF-7 HS578T, people's mammary gland normal cell system HS578BST, then utilizes NOTCH signal path detection reagent to detect the change of core NOTCH molecule in two kinds of clones.
MCF-7 HS578T used in the present invention, people's mammary gland normal cell system HS578BST purchased from American ATCC company, DMEM substratum, foetal calf serum purchased from American Gibco company, for extracting the RNAiso of total serum IgE, Reverse Transcriptase kit,
premix Ex Taq test kit is bought from TaKaRa.
Utilize reagent of the present invention to detect NOTCH signal path in cell and comprise following concrete steps:
One, MCF-7 HS578T, people's mammary gland normal cell system HS578BST cultivation with go down to posterity
For HS578T cell, in 60mm Tissue Culture Plate, add 1mL0.2% trypsin solution, shake gently pancreatin is fully contacted with cell, in 37 DEG C of carbonic acid gas incubators, hatch 2min, after Microscopic observation most cells floats, add 4mL containing the DMEM substratum of 10%FBS, blow even after add in 15mL centrifuge tube, the centrifugal 6min of 2000g, supernatant discarded, add corresponding proper volume nutrient solution according to cell category, repeatedly blow and beat for several times with 10mL transfer pipet, make cell free.Avoid in this process producing bubble, in order to avoid affect cell counting.Obtained cell suspension 20 μ L, in instillation cell counting count board, by the large lattice inner cell sum in cell counting metering corner four.The cell that during counting, only counting cells form is complete, the cell of accumulation calculates by 1 cell.Calculation formula is as follows:
Cell concn (individual/mL)=(4 large lattice total cellular score) × 10
4× extension rate/4.
5 × 10 are diluted to according to the corresponding nutrient solution cell suspension of cell category
6individual/mL, adds cell in 60mm Tissue Culture Dish, and every this cell diluent of plate 2mL, is placed in 37 DEG C, 5%CO
2cultivate in cell culture incubator, cell is generally adherent after 0.5h-1h, starts growth, become monolayer cell after 24h-48h after a few hours.
Two, the extraction of total serum IgE
1) CO is got
2be in cell one dish of logarithmic phase 60mm plate in incubator, after sopping up nutrient solution, after cleaning 2 times with aseptic 1 × PBS, add 1mL RNAiso at cell surface, repeatedly aspirate evenly with rifle head, proceed in sterile tube.
2) incubate at room temperature 5min, to dissolve nucleoprotein.Add 0.2mL phenol/chloroform (1:1), thermal agitation 15sec, incubated at room 2-3min.
3) 4 DEG C, the centrifugal 15min of 12000g, cell can produce layering.
4) supernatant liquid is gone in the centrifuge tube without RNase, add 0.5mL Virahol, put upside down mixing 3 times, incubate at room temperature 10min.
5) 4 DEG C, 12000g centrifugal 10min, RNA form bottom settlings.
6) supernatant is abandoned, with the 75% ethanol 1mL washing precipitation without RNase.4 DEG C, the centrifugal 5min of 7000g.
8) air drying RNA is about 5-10min.Should not be too dry.
9) with 30-50 μ L DEPC water dissolution RNA.
As shown in Figure 1, shown in Fig. 1 is extraction two kinds of cell total rnas to experimental result, and then carry out the result of electrophoresis, wherein 1 swimming lane represents HS578BST cell, and 2 swimming lanes are HS578T cell.
Three, the synthesis of cDNA
1) in the test tube of precooling, following reaction mixture is added:
Total serum IgE 1-5 μ g
oligo(dT)(0.5μg/μl) 1μL
12 μ L are settled to RNase-free water.
2) mix, centrifugal 3-5sec.
3) ice bath 30sec, centrifugal 3-5sec after 70 DEG C of effect 5min.
4) by test tube ice bath, following component is added:
5×Reaction buffer 4μL
Rnase Inhibiter(20U/μL) 1μL
dNTP mix(10mM) 2μL
5) mix gently, centrifugal 3-5sec.
6) 37 DEG C of water-bath 5min, add the M-MuLV Reverse Transcriptase (20U/ μ L) of 1 μ L, mixing.
7) 37 DEG C of effect 60min.Hatch 10min for 70 DEG C and terminate reaction, put and carry out downstream tests on ice.
Four, real-time fluorescence quantitative PCR
Getting synthetic cDNA is template, carries out real-time fluorescence quantitative PCR reaction according to following system:
The LightCycler480 instrument of Roche arranges following program: 95 DEG C of denaturations, 30 seconds; 95 DEG C of sex change, 5 seconds, 60 DEG C, 20 seconds, carry out 40 circulations.Solubility curve analyzes 95 DEG C, 15 seconds, to 65 DEG C, 1 minute, carries out gradient cooling according to 4.4 DEG C/sec.
(1) PCR detecting ADAM10 gene reacts primer, and its primer sequence is as follows:
5'-ATGGGAGGTCAGTATGGGAAT-3'；（SEQ ID No:1）
5'-ACTGCTCTTTTGGCACGC-3'；（SEQ ID No:2）
(2) PCR detecting ADAM17 gene reacts primer, and its primer sequence is as follows:
5'-ATGTTTCACGTTTGCAGTCTC-3'；（SEQ ID No:3）
5'-AGAAGCGATGATCTGCTACC-3'；（SEQ ID No:4）
(3) detect AES gene PCR react primer, its primer sequence is as follows:
5'-ACCCCAGCAACTCAAATTCA-3'；（SEQ ID No:5）
5'-AAGCCGTAGGACATCTCGTA-3'；（SEQ ID No:6）
(4) detect CBL gene PCR react primer, its primer sequence is as follows:
5'-TGGTGCGGTTGTGTCAGA-3'；（SEQ ID No:7）
5'-GGTAGGTATCTGGTAGCAGG-3'；（SEQ ID No:8）
(5) detect CCND1 gene PCR react primer, its primer sequence is as follows:
5'-GCTGCGAAGTGGAAACCAT-3'；（SEQ ID No:9）
5'-CCTCCTTCTGCACACATTTGA-3'；（SEQ ID No:10）
(6) detect CD44 gene PCR react primer, its primer sequence is as follows:
5'-CCCATCCCAGACGAAGACA-3'；（SEQ ID No:11）
5'-GCCATTTGTGTTGTTGTGTGA-3'；（SEQ ID No:12）
(7) PCR detecting CDKN1A gene reacts primer, and its primer sequence is as follows:
5'-TGTCCGTCAGAACCCATG-3'；（SEQ ID No:13）
5'-AAAGTCGAAGTTCCATCGCT-3'；（SEQ ID No:14）
(8) PCR detecting CFLAR gene reacts primer, and its primer sequence is as follows:
5'-AGAGTGAGGCGATTTGACCT-3'；（SEQ ID No:15）
5'-GTCCGAAACAAGGTGAGGGT-3'；（SEQ ID No:16）
(9) PCR detecting CHUK gene reacts primer, and its primer sequence is as follows:
5'-ATGAAGAAGTTGAACCATGCC-3'；（SEQ ID No:17）
5'-CCTCCAGAACAGTATTCCATTG-3'；（SEQ ID No:18）
(10) PCR detecting CTNNB1 gene reacts primer, and its primer sequence is as follows:
5'-ATGTCCAGCGTTTGGCTGA-3'；（SEQ ID No:19）
5'-TGGTCCTCGTCATTTAGCAGT-3'；（SEQ ID No:20）
(11) PCR detecting DLL1 gene reacts primer, and its primer sequence is as follows:
5'-TGTGACGAACACTACTACGG-3'；（SEQ ID No:21）
5'-AGCCAGGGTTGCACACTT-3';（SEQ ID No:22）
(12) PCR detecting DLL3 gene reacts primer, and its primer sequence is as follows:
5'-GCCAGAGCTTTTCCACTGATT-3'；（SEQ ID No:23）
5'-GCGTCAGGCCTAGAAACAAAA-3'；（SEQ ID No:24）
(13) PCR detecting DLL4 gene reacts primer, and its primer sequence is as follows:
5'-ACCTTCTCGCTCATCATCGA-3'；（SEQ ID No:25）
5'-CCTGGATGGCGATCTTGC-3'；（SEQ ID No:26）
(14) PCR detecting DTX1 gene reacts primer, and its primer sequence is as follows:
5'-CAGTCCATGCACCAGTTTC-3'；（SEQ ID No:27）
5'-TCCATATCGTAGGCCGTCCA-3'；（SEQ ID No:28）
(15) PCR detecting ERBB2 gene reacts primer, and its primer sequence is as follows:
5'-GCTCATCGCTCACAACCAAG-3'；（SEQ ID No:29）
5'-ACAGGGGTGGTATTGTTCAG-3'；（SEQ ID No:30）
(16) PCR detecting FOSL1 gene reacts primer, and its primer sequence is as follows:
5'-CAGGCGGAGACTGACAAACT-3'；（SEQ ID No:31）
5'-TCCTTCCGGGATTTTGCAGA-3'；（SEQ ID No:32）
(17) PCR detecting FZD1 gene reacts primer, and its primer sequence is as follows:
5'-ATCTTCTTGTCCGGCTGTTAC-3'；（SEQ ID No:33）
5'-GTCCTCGGCGAACTTGTCAT-3'；（SEQ ID No:34）
(18) PCR detecting FZD2 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCATCCTATCTCAGCTAC-3'；（SEQ ID No:35）
5'-CTGCATGTCTACCAAGTACGT-3'；（SEQ ID No:36）
(19) PCR detecting FZD3 gene reacts primer, and its primer sequence is as follows:
5'-GGATTGTTCTCGGGATTTCC-3'；（SEQ ID No:37）
5'-AGTGTGACACGTCCATATTCC-3'；（SEQ ID No:38）
(20) PCR detecting FZD4 gene reacts primer, and its primer sequence is as follows:
5'-GTCTTTCAGTCAAGAGACGCT-3'；（SEQ ID No:39）
5'-GTTGTGGTCGTTCTGTGGT-3'；（SEQ ID No:40）
(21) PCR detecting FZD6 gene reacts primer, and its primer sequence is as follows:
5'-GTGCAACTCTGTTCACATTCC-3'；（SEQ ID No:41）
5'-GCTATCGCCTAGCAAAAATCC-3'；（SEQ ID No:42）
(22) PCR detecting FZD7 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCAACGGCCTGATGT-3'；（SEQ ID No:43）
5'-AGGTGAGAACGGTAAAGAGC-3'；（SEQ ID No:44）
(23) PCR detecting GBP2 gene reacts primer, and its primer sequence is as follows:
5'-TTTCCAGCATTTGTGTGGAC-3'；（SEQ ID No:45）
5'-GGGAAGAACTTTCGGATGCA-3'；（SEQ ID No:46）
(24) PCR detecting Gli1 gene reacts primer, and its primer sequence is as follows:
5'-TTCCTACCAGAGTCCCAAG-3'；（SEQ ID No:47）
5'-CCCTATGTGAAGCCCTATT-3'；（SEQ ID No:48）
(25) PCR detecting GSK3B gene reacts primer, and its primer sequence is as follows:
5'-GGCAGCATGAAAGTTAGCAG-3'；（SEQ ID No:49）
5'-GGCGACCAGTTCTCCTGAAT-3'；（SEQ ID No:50）
(26) PCR detecting HES1 gene reacts primer, and its primer sequence is as follows:
5'-ACACGACACCGGATAAACCA-3'；（SEQ ID No:51）
5'-GCCGCGAGCTATCTTTCTTC-3'；（SEQ ID No:52）
(27) PCR detecting HEY1 gene reacts primer, and its primer sequence is as follows:
5'-ATCTGCTAAGCTAGAAAAAGCC-3'；（SEQ ID No:53）
5'-CGTCAAAGTAACCTTTCCCTCC-3'；（SEQ ID No:54）
(28) PCR detecting HEYL gene reacts primer, and its primer sequence is as follows:
5'-GGCTGCTTACGTGGCTGT-3'；（SEQ ID No:55）
5'-GACCCAGGAGTGGTAGAGCA-3'；（SEQ ID No:56）
(29) PCR detecting HOXB4 gene reacts primer, and its primer sequence is as follows:
5'-CGTGAGCACGGTAAACCC-3'；（SEQ ID No:57）
5'-CGAGCGGATCTTGGTGTT-3'；（SEQ ID No:58）
(30) PCR detecting HR gene reacts primer, and its primer sequence is as follows:
5'-CCCAGAGCATCCATGTGACT-3'；（SEQ ID No:59）
5'-TGCCCACCAGAGTTTAAGC-3'；（SEQ ID No:60）
(31) PCR detecting IFNG gene reacts primer, and its primer sequence is as follows:
5'-TCGGTAACTGACTTGAATGTCC-3'；（SEQ ID No:61）
5'-TCGCTTCCCTGTTTTAGCTG-3'；（SEQ ID No:62）
(32) PCR detecting IL17B gene reacts primer, and its primer sequence is as follows:
5'-GAGCCCCAAAAGCAAGAGGA-3'；（SEQ ID No:63）
5'-TGCGGGCATACGGTTTCAT-3'；（SEQ ID No:64）
(33) PCR detecting IL2RA gene reacts primer, and its primer sequence is as follows:
5'-GTGGGGACTGCTCACGTT-3'；（SEQ ID No:65）
5'-GGATCTCTGGCGGGTCAT-3'；（SEQ ID No:66）
(34) PCR detecting ITGA1 gene reacts primer, and its primer sequence is as follows:
5'-CTGGACATAGTCATAGTGCTGG-3'；（SEQ ID No:67）
5'-ACCTGTGTCTGTTTAGGACC-3'；（SEQ ID No:68）
(35) PCR detecting ITGA2 gene reacts primer, and its primer sequence is as follows:
5'-GGGAATCAGTATTACACAACGG-3'；（SEQ ID No:69）
5'-CCACAACATCTATGAGGGAAGG-3'；（SEQ ID No:70）
(36) PCR detecting ITGA3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGCTTGGAGTGACTGT-3'；（SEQ ID No:71）
5'-TCATTGCCTCGCACGTAG-3'；（SEQ ID No:72）
(37) PCR detecting ITGA4 gene reacts primer, and its primer sequence is as follows:
5'-GGTAGCCCTAATGGAGAACC-3'；（SEQ ID No:73）
5'-TATGCCCACAAGTCACGATG-3'；（SEQ ID No:74）
(38) PCR detecting ITGAV gene reacts primer, and its primer sequence is as follows:
5'-CCCCATTGTACCATTGGAGAA-3'；（SEQ ID No:75）
5'-TGGAGCATACTCAACAGTCTTT-3'；（SEQ ID No:76）
(39) PCR detecting ITGB1 gene reacts primer, and its primer sequence is as follows:
5'-CAAGAGAGCTGAAGACTATCCC-3'；（SEQ ID No:77）
5'-TGAAGTCCGAAGTAATCCTCC-3'；（SEQ ID No:78）
(40) PCR detecting ITGB3 gene reacts primer, and its primer sequence is as follows:
5'-GTGACCTGAAGGAGAATCTG-3'；（SEQ ID No:79）
5'-CCGGAGTGCAATCCTCTG-3'；（SEQ ID No:80）
(41) PCR detecting ITGB5 gene reacts primer, and its primer sequence is as follows:
5'-GGAAGTTCGGAAACAGAGGG-3'；（SEQ ID No:81）
5'-CTTTCGCCAGCCAATCTTCT-3'；（SEQ ID No:82）
(42) PCR detecting JAG1 gene reacts primer, and its primer sequence is as follows:
5'-GGGGCAACACCTTCAACCT-3'；（SEQ ID No:83）
5'-CCAGGCGAAACTGAAAGG-3'；（SEQ ID No:84）
(43) PCR detecting JAG2 gene reacts primer, and its primer sequence is as follows:
5'-TGGGCGGCAACTCCTTCT-3'；（SEQ ID No:85）
5'-GCCTCCACGATGAGGGTAA-3'；（SEQ ID No:86）
(44) PCR detecting KAT2B gene reacts primer, and its primer sequence is as follows:
5'-GAAAAACCCTAACCCCTCAC-3'；（SEQ ID No:87）
5'-CCTTGTGGACACAGGTAAAGAG-3'；（SEQ ID No:88）
(45) PCR detecting KRT1 gene reacts primer, and its primer sequence is as follows:
5'-GGTGCTTATATGACCAAGGTG-3'；（SEQ ID No:89）
5'-ATGCTGTCCAGGTCGAGAC-3'；（SEQ ID No:90）
(46) PCR detecting LFNG gene reacts primer, and its primer sequence is as follows:
5'-GTCAGCGAGAACAAGGTG-3'；（SEQ ID No:91）
5'-GATCCGCTCAGCCGTATTCA-3'；（SEQ ID No:92）
(47) PCR detecting LMO2 gene reacts primer, and its primer sequence is as follows:
5'-GACAAGCGGATTCGTGCCTA-3'；（SEQ ID No:93）
5'-AGTTGATGAGGAGGTATCTGTC-3'；（SEQ ID No:94）
(48) PCR detecting MAP2K7 gene reacts primer, and its primer sequence is as follows:
5'-TGCTTTGGGACGTTCATCAC-3'；（SEQ ID No:95）
5'-CCACTGTCATCTTGCCCAGA-3'；（SEQ ID No:96）
(49) PCR detecting MFNG gene reacts primer, and its primer sequence is as follows:
5'-TGCTGAGTTCGACACCTTCT-3'；（SEQ ID No:97）
5'-GCCCTTGGGTTCACATAGTT-3'；（SEQ ID No:98）
(50) PCR detecting MMP7 gene reacts primer, and its primer sequence is as follows:
5'-GAGTGAGCTACAGTGGGAAC-3'；（SEQ ID No:99）
5'-CTATGACGCGGGAGTTTAACA-3'；（SEQ ID No:100）
(51) PCR detecting MYCL1 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCCCAAACCTGTCA-3'；（SEQ ID No:101）
5'-GGCCTTGCTTAGGATCACTACT-3'；（SEQ ID No:102）
(52) PCR detecting NCOR2 gene reacts primer, and its primer sequence is as follows:
5'-TGCAGATCATCTACGACGAG-3'；（SEQ ID No:103）
5'-TCCGCATCGCCTGGTTTATT-3'；（SEQ ID No:104）
(53) PCR detecting NESTIN gene reacts primer, and its primer sequence is as follows:
5'-AGCCCTGACCACTCCAGTTTA-3'；（SEQ ID No:105）
5'-CCCTCTATGGCTGTTTCTTTCTC-3'；（SEQ ID No:106）
(54) PCR detecting NEURL gene reacts primer, and its primer sequence is as follows:
5'-GTGTCTTCCACCGCATCAA-3'；（SEQ ID No:107）
5'-ACCAGCTCGCTATCAAGCA-3'；（SEQ ID No:108）
(55) PCR detecting NFKB1 gene reacts primer, and its primer sequence is as follows:
5'-GAAGCACGAATGACAGAGG-3'；（SEQ ID No:109）
5'-GCTTGGCGGATTAGCTCTTT-3'；（SEQ ID No:110）
(56) PCR detecting NFKB2 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAGAGTTGCTACAACCC-3'；（SEQ ID No:111）
5'-CTGTTCCACGATCACCAGGT-3'；（SEQ ID No:112）
(57) PCR detecting Notch1 gene reacts primer, and its primer sequence is as follows:
5'-ATCGACGATTGTCCAGGAAAC-3'；（SEQ ID No:113）
5'-TACTGACCTGTCCACTCTGGC-3'；（SEQ ID No:114）
(58) PCR detecting NOTCH2 gene reacts primer, and its primer sequence is as follows:
5'-AAGCAGAGTCCCAGTGCCT-3'；（SEQ ID No:115）
5'-CAGGGGGCACTGACAGTAA-3'；（SEQ ID No:116）
(59) PCR detecting NOTCH2NL gene reacts primer, and its primer sequence is as follows:
5'-TGTGACAGCCTGTATGTGC-3'；（SEQ ID No:117）
5'-TGAGCAGCTTAGGAAAGATTG-3'；（SEQ ID No:118）
(60) PCR detecting NOTCH3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGACGAGTGCTCTATC-3'；（SEQ ID No:119）
5'-AATGTCCACCTCGCAATAG-3'；（SEQ ID No:120）
(61) PCR detecting NOTCH4 gene reacts primer, and its primer sequence is as follows:
5'-CTAGGGGCTCTTCTCGTCC-3'；（SEQ ID No:121）
5'-CAACTTCTGCCTTTGGCTT-3'；（SEQ ID No:122）
(62) PCR detecting NR4A2 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCTTGCTTGTACCAAA-3'；（SEQ ID No:123）
5'-GGCTTGTAGTAAACCGACCC-3'；（SEQ ID No:124）
(63) PCR detecting NUMB gene reacts primer, and its primer sequence is as follows:
5'-TCAGCAGATGGACTCAGAGT-3'；（SEQ ID No:125）
5'-AGGCTCTATCAAAGTTCCTGTC-3'；（SEQ ID No:126）
(64) PCR detecting PAX5 gene reacts primer, and its primer sequence is as follows:
5'-ACTTGCTCATCAAGGTGTCA-3'；（SEQ ID No:127）
5'-TCCTCCAATTACCCCAGGCT-3'；（SEQ ID No:128）
(65) PCR detecting PDPK1 gene reacts primer, and its primer sequence is as follows:
5'-ACGGAGAAGTCCGCCTGTA-3'；（SEQ ID No:129）
5'-CTCGTTTCCAGCTCGGAAT-3'；（SEQ ID No:130）
(66) PCR detecting POFUT1 gene reacts primer, and its primer sequence is as follows:
5'-AACCAGGCCGATCACTTCTT-3'；（SEQ ID No:131）
5'-GTTGGTGAAAGGAGGCTTGT-3'；（SEQ ID No:132）
(67) PCR detecting PPARG gene reacts primer, and its primer sequence is as follows:
5'-GGGATCAGCTCCGTGGATC-3'；（SEQ ID No:133）
5'-TGCACTTTGGTACTCTTGAAGT-3'；（SEQ ID No:134）
(68) PCR detecting PSEN1 gene reacts primer, and its primer sequence is as follows:
5'-GACGACCCCAGGGTAACT-3'；（SEQ ID No:135）
5'-ACTGACTTAATGGTAGCCACG-3'；（SEQ ID No:136）
(69) PCR detecting PSEN2 gene reacts primer, and its primer sequence is as follows:
5'-ACCGCTATGTCTGTAGTG-3'；（SEQ ID No:137）
5'-CGCTCCGTATTTGAGGGTCA-3'；（SEQ ID No:138）
(70) PCR detecting PSENEN gene reacts primer, and its primer sequence is as follows:
5'-CTGGAGCGAGTGTCCAATGA-3'；（SEQ ID No:139）
5'-GCGCCAGACATAGCCTTTGA-3'；（SEQ ID No:140）
(71) PCR detecting RFNG gene reacts primer, and its primer sequence is as follows:
5'-CGCCAGCAGACGTTTATCTT-3'；（SEQ ID No:141）
5'-CCGAGCAGTTGGTGTTGAT-3'；（SEQ ID No:142）
(72) PCR detecting RUNX1 gene reacts primer, and its primer sequence is as follows:
5'-CTGCCCATCGCTTTCAAGG-3'；（SEQ ID No:143）
5'-GCCGAGTAGTTTTCATCATTGC-3'；（SEQ ID No:144）
(73) PCR detecting SEL1L gene reacts primer, and its primer sequence is as follows:
5'-AAACCAGCTTTGACCGCCA-3'；（SEQ ID No:145）
5'-GTCATAGGTTGTAGCACACCA-3'；（SEQ ID No:146）
(74) PCR detecting SH2D1A gene reacts primer, and its primer sequence is as follows:
5'-GACAGCACCTGGGGTACATA-3'；（SEQ ID No:147）
5'-TGCAGAGGTATTACAATGCCTT-3'；（SEQ ID No:148）
(75) PCR detecting SHH gene reacts primer, and its primer sequence is as follows:
5'-CTCGCTGCTGGTATGCTC-3'；（SEQ ID No:149）
5'-ATCGCTCGGAGTTTCTGGAG-3'；（SEQ ID No:150）
(76) PCR detecting Smo gene reacts primer, and its primer sequence is as follows:
5'-GTTCTCCATCAAGAGCAACCA-3'；（SEQ ID No:151）
5'-CGATTCTTGATCTCACAGTCAG-3'；（SEQ ID No:152）
(77) PCR detecting SNW1 gene reacts primer, and its primer sequence is as follows:
5'-GGATACCGGAAAGGCTGGAT-3'；（SEQ ID No:153）
5'-TGACTGTCCTTGTCGAGCAA-3'；（SEQ ID No:154）
(78) PCR detecting STAT6 gene reacts primer, and its primer sequence is as follows:
5'-GTTCCGCCACTTGCCAAT-3'；（SEQ ID No:155）
5'-GTTCCGCCACTTGCCAAT-3'；（SEQ ID No:156）
(79) PCR detecting SUFU gene reacts primer, and its primer sequence is as follows:
5'-CCAGGTTACCGCTATCGTCA-3'；（SEQ ID No:157）
5'-CGAAGCTGATGTAGTGCCAG-3'；（SEQ ID No:158）
(80) PCR detecting TEAD1 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAAAGGATGAGTGACTCTG-3'；（SEQ ID No:159）
5'-TCCCACATGGTGGATAGATAG-3'；（SEQ ID No:160）
(81) PCR detecting TLE1 gene reacts primer, and its primer sequence is as follows:
5'-GAGTCCCTGGACCGGATTAA-3'；（SEQ ID No:161）
5'-AATACATCACATAGTGCCTCTG-3'；（SEQ ID No:162）
(82) PCR detecting WISP1 gene reacts primer, and its primer sequence is as follows:
5'-TGCTGTAAGATGTGCGCTCA-3'；（SEQ ID No:163）
5'-ACACTCCTATTGCGTACCTC-3'；（SEQ ID No:164）
(83) PCR detecting WNT11 gene reacts primer, and its primer sequence is as follows:
5'-GGAGTCGGCCTTCGTGTAT-3'；（SEQ ID No:165）
5'-GCTGAGGTTGTCCGCACA-3'；（SEQ ID No:166）
(84) PCR detecting ZIC2 gene reacts primer, and its primer sequence is as follows:
5'-CACCTCCGATAAGCCCTATC-3'；（SEQ ID No:167）
5'-GGCGTGGACGACTCATAG-3'；（SEQ ID No:168）
(85) PCR detecting GAPDH gene reacts primer, and its primer sequence is as follows:
5'-ACTGCCACCCAGAAGAC-3'；（SEQ ID No:169）
5'-GCTCAGTGTAGCCCAGGA-3'；（SEQ ID No:170）
(86) PCR detecting ACTB gene reacts primer, and its primer sequence is as follows:
5'-CATGTACGTTGCTATCCAGG-3'；（SEQ ID No:171）
5'-CTCCTTAATGTCACGCACGA-3'；（SEQ ID No:172）
(87) PCR detecting B2M gene reacts primer, and its primer sequence is as follows:
5'-GAGGCTATCCAGCGTACTCC-3'；（SEQ ID No:173）
5'-CGGCAGGCATACTCATCTTT-3'（SEQ ID No:174）。
Above-mentioned primer is placed in the enterprising performing PCR reaction of 96 orifice plate, and wherein often pair of primer is placed in arbitrary hole, altogether 87 pairs of primers is placed in 87 holes.
As shown in Figure 2, shown in Fig. 2 is amplification curve after the real-time fluorescence quantitative PCR carrying out NOTCH signal path core gene to experimental result, and this result can see that goal gene amplification is good, is conducive to the analysis and research in downstream.
Five, data analysis
The Ct value of each gene is utilized to calculate the differential expression of core NOTCH gene, as shown in Figure 3 and Figure 4.Fig. 3 shows the result of HS578BST and HS578T after solubility curve is analyzed, and the solubility curve of each gene is unimodal, illustrates that PCR primer is single, does not have the appearance at the assorted peaks such as primer dimer, and the double-stranded DNA of amplification is goal gene.Fig. 4 is the differential gene expression result obtained after the PCR result of comparative analysis HS578BST and HS578T, and this result has filtered out affects the core Notch signal path gene that development occurs mammary cancer.
The present invention will relate to molecule closely-related with Notch signal path, this core element concentrates on a flat board, by doing a real-time fluorescence quantitative PCR reaction, the survival condition of reacting cells, contrast with normal mammary gland cell, mammary cancer, inquire into the possible approaches of Notch signal path in tumour/normal cell, for the regulation and control studying key protein provide the most direct evidence; The present invention finds Notch signal path associated molecule fast and accurately from transcriptional level, for the Mechanism Discussion etc. of screening anticancer medicine, new targeted drug provides strong instrument.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (5)
1. detect a reagent for cell NOTCH signal path, it is characterized in that it comprises following primer:
(1) PCR detecting ADAM10 gene reacts primer, and its primer sequence is as follows:
5'- ATGGGAGGTCAGTATGGGAAT-3'；
5'- ACTGCTCTTTTGGCACGC-3'；
(2) PCR detecting ADAM17 gene reacts primer, and its primer sequence is as follows:
5'- ATGTTTCACGTTTGCAGTCTC-3'；
5'- AGAAGCGATGATCTGCTACC-3'；
(3) detect AES gene PCR react primer, its primer sequence is as follows:
5'- ACCCCAGCAACTCAAATTCA-3'；
5'- AAGCCGTAGGACATCTCGTA-3'；
(4) detect CBL gene PCR react primer, its primer sequence is as follows:
5'- TGGTGCGGTTGTGTCAGA-3'；
5'- GGTAGGTATCTGGTAGCAGG-3'；
(5) detect CCND1 gene PCR react primer, its primer sequence is as follows:
5'- GCTGCGAAGTGGAAACCAT-3'；
5'- CCTCCTTCTGCACACATTTGA-3'；
(6) detect CD44 gene PCR react primer, its primer sequence is as follows:
5'- CCCATCCCAGACGAAGACA -3'；
5'- GCCATTTGTGTTGTTGTGTGA -3'；
(7) PCR detecting CDKN1A gene reacts primer, and its primer sequence is as follows:
5'- TGTCCGTCAGAACCCATG-3'；
5'- AAAGTCGAAGTTCCATCGCT-3'；
(8) PCR detecting CFLAR gene reacts primer, and its primer sequence is as follows:
5'- AGAGTGAGGCGATTTGACCT-3'；
5'- GTCCGAAACAAGGTGAGGGT-3'；
(9) PCR detecting CHUK gene reacts primer, and its primer sequence is as follows:
5'- ATGAAGAAGTTGAACCATGCC-3'；
5'- CCTCCAGAACAGTATTCCATTG-3'；
(10) PCR detecting CTNNB1 gene reacts primer, and its primer sequence is as follows:
5'- ATGTCCAGCGTTTGGCTGA-3'；
5'- TGGTCCTCGTCATTTAGCAGT-3'；
(11) PCR detecting DLL1 gene reacts primer, and its primer sequence is as follows:
5'- TGTGACGAACACTACTACGG-3'；
5'- AGCCAGGGTTGCACACTT-3'；
(12) PCR detecting DLL3 gene reacts primer, and its primer sequence is as follows:
5'- GCCAGAGCTTTTCCACTGATT -3'；
5'- GCGTCAGGCCTAGAAACAAAA -3'；
(13) PCR detecting DLL4 gene reacts primer, and its primer sequence is as follows:
5'- ACCTTCTCGCTCATCATCGA -3'；
5'- CCTGGATGGCGATCTTGC-3'；
(14) PCR detecting DTX1 gene reacts primer, and its primer sequence is as follows:
5'- CAGTCCATGCACCAGTTTC-3'；
5'- TCCATATCGTAGGCCGTCCA-3'；
(15) PCR detecting ERBB2 gene reacts primer, and its primer sequence is as follows:
5'- GCTCATCGCTCACAACCAAG-3'；
5'- ACAGGGGTGGTATTGTTCAG-3'；
(16) PCR detecting FOSL1 gene reacts primer, and its primer sequence is as follows:
5'- CAGGCGGAGACTGACAAACT-3'；
5'- TCCTTCCGGGATTTTGCAGA-3'；
(17) PCR detecting FZD1 gene reacts primer, and its primer sequence is as follows:
5'- ATCTTCTTGTCCGGCTGTTAC-3'；
5'- GTCCTCGGCGAACTTGTCAT-3'；
(18) PCR detecting FZD2 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCATCCTATCTCAGCTAC-3'；
5'-CTGCATGTCTACCAAGTACGT-3'；
(19) PCR detecting FZD3 gene reacts primer, and its primer sequence is as follows:
5'-GGATTGTTCTCGGGATTTCC-3'；
5'- AGTGTGACACGTCCATATTCC-3'；
(20) PCR detecting FZD4 gene reacts primer, and its primer sequence is as follows:
5'- GTCTTTCAGTCAAGAGACGCT-3'；
5'- GTTGTGGTCGTTCTGTGGT-3'；
(21) PCR detecting FZD6 gene reacts primer, and its primer sequence is as follows:
5'-GTGCAACTCTGTTCACATTCC-3'；
5'-GCTATCGCCTAGCAAAAATCC-3'；
(22) PCR detecting FZD7 gene reacts primer, and its primer sequence is as follows:
5'-GTGCCAACGGCCTGATGT-3'；
5'-AGGTGAGAACGGTAAAGAGC-3'；
(23) PCR detecting GBP2 gene reacts primer, and its primer sequence is as follows:
5'- TTTCCAGCATTTGTGTGGAC-3'；
5'- GGGAAGAACTTTCGGATGCA-3'；
(24) PCR detecting Gli1 gene reacts primer, and its primer sequence is as follows:
5'- TTCCTACCAGAGTCCCAAG-3'；
5'- CCCTATGTGAAGCCCTATT-3'；
(25) PCR detecting GSK3B gene reacts primer, and its primer sequence is as follows:
5'- GGCAGCATGAAAGTTAGCAG-3'；
5'- GGCGACCAGTTCTCCTGAAT-3'；
(26) PCR detecting HES1 gene reacts primer, and its primer sequence is as follows:
5'- ACACGACACCGGATAAACCA-3'；
5'- GCCGCGAGCTATCTTTCTTC -3'；
(27) PCR detecting HEY1 gene reacts primer, and its primer sequence is as follows:
5'- ATCTGCTAAGCTAGAAAAAGCC -3'；
5'- CGTCAAAGTAACCTTTCCCTCC-3'；
(28) PCR detecting HEYL gene reacts primer, and its primer sequence is as follows:
5'- GGCTGCTTACGTGGCTGT-3'；
5'- GACCCAGGAGTGGTAGAGCA-3'；
(29) PCR detecting HOXB4 gene reacts primer, and its primer sequence is as follows:
5'-CGTGAGCACGGTAAACCC -3'；
5'-CGAGCGGATCTTGGTGTT -3'；
(30) PCR detecting HR gene reacts primer, and its primer sequence is as follows:
5'- CCCAGAGCATCCATGTGACT -3'；
5'- TGCCCACCAGAGTTTAAGC -3'；
(31) PCR detecting IFNG gene reacts primer, and its primer sequence is as follows:
5'- TCGGTAACTGACTTGAATGTCC -3'；
5'- TCGCTTCCCTGTTTTAGCTG -3'；
(32) PCR detecting IL17B gene reacts primer, and its primer sequence is as follows:
5'- GAGCCCCAAAAGCAAGAGGA-3'；
5'- TGCGGGCATACGGTTTCAT-3'；
(33) PCR detecting IL2RA gene reacts primer, and its primer sequence is as follows:
5'- GTGGGGACTGCTCACGTT -3'；
5'- GGATCTCTGGCGGGTCAT -3'；
(34) PCR detecting ITGA1 gene reacts primer, and its primer sequence is as follows:
5'-CTGGACATAGTCATAGTGCTGG -3'；
5'-ACCTGTGTCTGTTTAGGACC -3'；
(35) PCR detecting ITGA2 gene reacts primer, and its primer sequence is as follows:
5'- GGGAATCAGTATTACACAACGG -3'；
5'- CCACAACATCTATGAGGGAAGG -3'；
(36) PCR detecting ITGA3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGCTTGGAGTGACTGT -3'；
5'-TCATTGCCTCGCACGTAG -3'；
(37) PCR detecting ITGA4 gene reacts primer, and its primer sequence is as follows:
5'-GGTAGCCCTAATGGAGAACC-3'；
5'-TATGCCCACAAGTCACGATG -3'；
(38) PCR detecting ITGAV gene reacts primer, and its primer sequence is as follows:
5'-CCCCATTGTACCATTGGAGAA -3'；
5'-TGGAGCATACTCAACAGTCTTT -3'；
(39) PCR detecting ITGB1 gene reacts primer, and its primer sequence is as follows:
5'-CAAGAGAGCTGAAGACTATCCC -3'；
5'-TGAAGTCCGAAGTAATCCTCC -3'；
(40) PCR detecting ITGB3 gene reacts primer, and its primer sequence is as follows:
5'-GTGACCTGAAGGAGAATCTG -3'；
5'-CCGGAGTGCAATCCTCTG -3'；
(41) PCR detecting ITGB5 gene reacts primer, and its primer sequence is as follows:
5'-GGAAGTTCGGAAACAGAGGG -3'；
5'-CTTTCGCCAGCCAATCTTCT -3'；
(42) PCR detecting JAG1 gene reacts primer, and its primer sequence is as follows:
5'-GGGGCAACACCTTCAACCT -3'；
5'-CCAGGCGAAACTGAAAGG -3'；
(43) PCR detecting JAG2 gene reacts primer, and its primer sequence is as follows:
5'-TGGGCGGCAACTCCTTCT -3'；
5'-GCCTCCACGATGAGGGTAA -3'；
(44) PCR detecting KAT2B gene reacts primer, and its primer sequence is as follows:
5'-GAAAAACCCTAACCCCTCAC -3'；
5'-CCTTGTGGACACAGGTAAAGAG -3'；
(45) PCR detecting KRT1 gene reacts primer, and its primer sequence is as follows:
5'-GGTGCTTATATGACCAAGGTG -3'；
5'-ATGCTGTCCAGGTCGAGAC -3'；
(46) PCR detecting LFNG gene reacts primer, and its primer sequence is as follows:
5'-GTCAGCGAGAACAAGGTG -3'；
5'-GATCCGCTCAGCCGTATTCA -3'；
(47) PCR detecting LMO2 gene reacts primer, and its primer sequence is as follows:
5'-GACAAGCGGATTCGTGCCTA -3'；
5'-AGTTGATGAGGAGGTATCTGTC -3'；
(48) PCR detecting MAP2K7 gene reacts primer, and its primer sequence is as follows:
5'-TGCTTTGGGACGTTCATCAC -3'；
5'-CCACTGTCATCTTGCCCAGA -3'；
(49) PCR detecting MFNG gene reacts primer, and its primer sequence is as follows:
5'-TGCTGAGTTCGACACCTTCT -3'；
5'-GCCCTTGGGTTCACATAGTT -3'；
(50) PCR detecting MMP7 gene reacts primer, and its primer sequence is as follows:
5'-GAGTGAGCTACAGTGGGAAC -3'；
5'-CTATGACGCGGGAGTTTAACA -3'；
(51) PCR detecting MYCL1 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCCCAAACCTGTCA -3'；
5'-GGCCTTGCTTAGGATCACTACT -3'；
(52) PCR detecting NCOR2 gene reacts primer, and its primer sequence is as follows:
5'-TGCAGATCATCTACGACGAG -3'；
5'-TCCGCATCGCCTGGTTTATT -3'；
(53) PCR detecting NESTIN gene reacts primer, and its primer sequence is as follows:
5'-AGCCCTGACCACTCCAGTTTA-3'；
5'- CCCTCTATGGCTGTTTCTTTCTC -3'；
(54) PCR detecting NEURL gene reacts primer, and its primer sequence is as follows:
5'-GTGTCTTCCACCGCATCAA -3'；
5'-ACCAGCTCGCTATCAAGCA -3'；
(55) PCR detecting NFKB1 gene reacts primer, and its primer sequence is as follows:
5'-GAAGCACGAATGACAGAGG -3'；
5'-GCTTGGCGGATTAGCTCTTT -3'；
(56) PCR detecting NFKB2 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAGAGTTGCTACAACCC -3'；
5'-CTGTTCCACGATCACCAGGT -3'；
(57) PCR detecting Notch1 gene reacts primer, and its primer sequence is as follows:
5'-ATCGACGATTGTCCAGGAAAC -3'；
5'-TACTGACCTGTCCACTCTGGC -3'；
(58) PCR detecting NOTCH2 gene reacts primer, and its primer sequence is as follows:
5'-AAGCAGAGTCCCAGTGCCT -3'；
5'-CAGGGGGCACTGACAGTAA -3'；
(59) PCR detecting NOTCH2NL gene reacts primer, and its primer sequence is as follows:
5'-TGTGACAGCCTGTATGTGC -3'；
5'-TGAGCAGCTTAGGAAAGATTG -3'；
(60) PCR detecting NOTCH3 gene reacts primer, and its primer sequence is as follows:
5'-TGTGGACGAGTGCTCTATC -3'；
5'-AATGTCCACCTCGCAATAG -3'；
(61) PCR detecting NOTCH4 gene reacts primer, and its primer sequence is as follows:
5'-CTAGGGGCTCTTCTCGTCC -3'；
5'-CAACTTCTGCCTTTGGCTT -3'；
(62) PCR detecting NR4A2 gene reacts primer, and its primer sequence is as follows:
5'-GCCACCTTGCTTGTACCAAA -3'；
5'-GGCTTGTAGTAAACCGACCC -3'；
(63) PCR detecting NUMB gene reacts primer, and its primer sequence is as follows:
5'-TCAGCAGATGGACTCAGAGT -3'；
5'-AGGCTCTATCAAAGTTCCTGTC -3'；
(64) PCR detecting PAX5 gene reacts primer, and its primer sequence is as follows:
5'-ACTTGCTCATCAAGGTGTCA -3'；
5'-TCCTCCAATTACCCCAGGCT -3'；
(65) PCR detecting PDPK1 gene reacts primer, and its primer sequence is as follows:
5'-ACGGAGAAGTCCGCCTGTA -3'；
5'-CTCGTTTCCAGCTCGGAAT -3'；
(66) PCR detecting POFUT1 gene reacts primer, and its primer sequence is as follows:
5'-AACCAGGCCGATCACTTCTT -3'；
5'-GTTGGTGAAAGGAGGCTTGT -3'；
(67) PCR detecting PPARG gene reacts primer, and its primer sequence is as follows:
5'-GGGATCAGCTCCGTGGATC -3'；
5'-TGCACTTTGGTACTCTTGAAGT -3'；
(68) PCR detecting PSEN1 gene reacts primer, and its primer sequence is as follows:
5'-GACGACCCCAGGGTAACT -3'；
5'-ACTGACTTAATGGTAGCCACG -3'；
(69) PCR detecting PSEN2 gene reacts primer, and its primer sequence is as follows:
5'-ACCGCTATGTCTGTAGTG -3'；
5'-CGCTCCGTATTTGAGGGTCA -3'；
(70) PCR detecting PSENEN gene reacts primer, and its primer sequence is as follows:
5'-CTGGAGCGAGTGTCCAATGA -3'；
5'-GCGCCAGACATAGCCTTTGA -3'；
(71) PCR detecting RFNG gene reacts primer, and its primer sequence is as follows:
5'-CGCCAGCAGACGTTTATCTT -3'；
5'-CCGAGCAGTTGGTGTTGAT -3'；
(72) PCR detecting RUNX1 gene reacts primer, and its primer sequence is as follows:
5'-CTGCCCATCGCTTTCAAGG -3'；
5'-GCCGAGTAGTTTTCATCATTGC -3'；
(73) PCR detecting SEL1L gene reacts primer, and its primer sequence is as follows:
5'-AAACCAGCTTTGACCGCCA -3'；
5'-GTCATAGGTTGTAGCACACCA -3'；
(74) PCR detecting SH2D1A gene reacts primer, and its primer sequence is as follows:
5'- GACAGCACCTGGGGTACATA -3'；
5'- TGCAGAGGTATTACAATGCCTT -3'；
(75) PCR detecting SHH gene reacts primer, and its primer sequence is as follows:
5'-CTCGCTGCTGGTATGCTC -3'；
5'-ATCGCTCGGAGTTTCTGGAG -3'；
(76) PCR detecting Smo gene reacts primer, and its primer sequence is as follows:
5'-GTTCTCCATCAAGAGCAACCA -3'；
5'-CGATTCTTGATCTCACAGTCAG -3'；
(77) PCR detecting SNW1 gene reacts primer, and its primer sequence is as follows:
5'-GGATACCGGAAAGGCTGGAT -3'；
5'-TGACTGTCCTTGTCGAGCAA -3'；
(78) PCR detecting STAT6 gene reacts primer, and its primer sequence is as follows:
5'-GTTCCGCCACTTGCCAAT -3'；
5'-GTTCCGCCACTTGCCAAT -3'；
(79) PCR detecting SUFU gene reacts primer, and its primer sequence is as follows:
5'-CCAGGTTACCGCTATCGTCA -3'；
5'-CGAAGCTGATGTAGTGCCAG -3'；
(80) PCR detecting TEAD1 gene reacts primer, and its primer sequence is as follows:
5'-ATGGAAAGGATGAGTGACTCTG -3'；
5'-TCCCACATGGTGGATAGATAG -3'；
(81) PCR detecting TLE1 gene reacts primer, and its primer sequence is as follows:
5'-GAGTCCCTGGACCGGATTAA-3'；
5'-AATACATCACATAGTGCCTCTG-3'；
(82) PCR detecting WISP1 gene reacts primer, and its primer sequence is as follows:
5'-TGCTGTAAGATGTGCGCTCA -3'；
5'-ACACTCCTATTGCGTACCTC-3'；
(83) PCR detecting WNT11 gene reacts primer, and its primer sequence is as follows:
5'-GGAGTCGGCCTTCGTGTAT -3'；
5'-GCTGAGGTTGTCCGCACA -3'；
(84) PCR detecting ZIC2 gene reacts primer, and its primer sequence is as follows:
5'- CACCTCCGATAAGCCCTATC -3'；
5'- GGCGTGGACGACTCATAG -3'。
2. the reagent of detection cell NOTCH signal path according to claim 1, is characterized in that: the PCR of described reagent also containing internal reference regulatory gene GAPDH, ACTB, B2M reacts primer.
3. the reagent of detection cell NOTCH signal path according to claim 2, is characterized in that:
The PCR detecting GAPDH gene reacts primer, and its primer sequence is as follows:
5'- ACTGCCACCCAGAAGAC -3'；
5'-GCTCAGTGTAGCCCAGGA-3'；
The PCR detecting ACTB gene reacts primer, and its primer sequence is as follows:
5'-CATGTACGTTGCTATCCAGG -3'；
5'-CTCCTTAATGTCACGCACGA-3'；
The PCR detecting B2M gene reacts primer, and its primer sequence is as follows:
5'-GAGGCTATCCAGCGTACTCC-3'；
5'- CGGCAGGCATACTCATCTTT-3'。
4. reagent according to claim 1 detects the application in the detection reagent of cancer cells in preparation.
5. reagent according to claim 4 detects the application in the detection reagent of cancer cells in preparation, it is characterized in that: described cancer cells is breast cancer cell, lung carcinoma cell and liver cancer cell.
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| CN102226177A (en) * | 2011-04-25 | 2011-10-26 | 周蒙滔 | Fluorescent quantitative detection kit for Notch pathway key molecule |
| CN103882138B (en) * | 2014-04-04 | 2015-09-09 | 上海赛安生物医药科技有限公司 | A kind of multiple gene detection kit relevant to anti-tumor drug |
| CN104498611B (en) * | 2014-12-22 | 2016-10-19 | 西北农林科技大学 | The RFLP method in detection cattle Notch1 gene SNP site and test kit |
| CN107227358A (en) * | 2017-06-16 | 2017-10-03 | 首都医科大学附属北京友谊医院 | Purposes of the NUMB in postmenopausal women's diagnosis of primary osteoporosis or prognosis |
| CN107475376A (en) * | 2017-08-02 | 2017-12-15 | 南方医科大学珠江医院 | Predict label and kit of the ED-SCLC to chemotherapy drug susceptibility |
| CN109735540B (en) * | 2019-01-22 | 2022-05-31 | 南京鼓楼医院 | SH2D1A gene, sgRNA and application thereof |
| CN109833469A (en) * | 2019-03-19 | 2019-06-04 | 大连医科大学 | PoFUT1 is preparing the application in placenta angiogenesis drug and RhoA signal path activator drug |
| CN111705130B (en) * | 2020-06-03 | 2022-07-12 | 广州康立明生物科技股份有限公司 | Gene marker combination and application thereof |
| CN111676287B (en) * | 2020-06-03 | 2022-04-29 | 广州康立明生物科技股份有限公司 | Gene marker combination and application thereof |
| CN111850122A (en) * | 2020-07-13 | 2020-10-30 | 青岛大学附属医院 | PCR detection reagent for detecting liver cancer core signal molecule and application thereof |
| CN111690751A (en) * | 2020-07-13 | 2020-09-22 | 青岛大学附属医院 | PCR reagent for detecting tumor drug target and application thereof |
| CN111850123A (en) * | 2020-07-13 | 2020-10-30 | 青岛大学附属医院 | PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof |
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| CN101878223A (en) * | 2007-10-05 | 2010-11-03 | 特洛伊科技有限公司 | Methods of treating cancer using NOTCH pathway inhibitors |
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| CN101878223A (en) * | 2007-10-05 | 2010-11-03 | 特洛伊科技有限公司 | Methods of treating cancer using NOTCH pathway inhibitors |
| CN102085372A (en) * | 2009-12-04 | 2011-06-08 | 中国医学科学院基础医学研究所 | Application of Notch pathway inhibitor in treatment of tumors caused by activation of mTOR |
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