CN111808958A - PCR reagent for detecting apoptosis signal path and application thereof - Google Patents

PCR reagent for detecting apoptosis signal path and application thereof Download PDF

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CN111808958A
CN111808958A CN202010666818.2A CN202010666818A CN111808958A CN 111808958 A CN111808958 A CN 111808958A CN 202010666818 A CN202010666818 A CN 202010666818A CN 111808958 A CN111808958 A CN 111808958A
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detecting
gene
pcr reaction
following primer
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仇静
于江波
袁荣涛
唐永平
陈正岗
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Qingdao Municipal Hospital
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Qingdao Municipal Hospital
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Abstract

The PCR reagent comprises PCR reaction primers for detecting related genes such as ABL1 gene, AIFM1 gene, AKT1 gene, APAF1 gene, BAD gene and the like and internal reference genes. The invention concentrates the signal molecules related to apoptosis on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, compares the survival state with normal cells and oral squamous cancer cell lines, discusses the possible ways of an apoptosis signal path in oral tumor/normal cells, and provides the most direct evidence for researching the regulation and control of key proteins; the invention can quickly and accurately find the apoptosis core signal channel related molecules from the transcription level, and provides a powerful tool for screening anti-cancer drugs, researching the mechanism of new targeted drugs and the like.

Description

PCR reagent for detecting apoptosis signal path and application thereof
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a PCR reagent for detecting an apoptosis signal pathway and application thereof.
Background
The apoptosis signal pathway is an extremely important signal transduction pathway existing in the organism, and plays an important role in the regulation of tumor cells in particular. At present, apoptosis is generally caused by physiological or pathological factors. During apoptosis, the cells shrink and the DNA is degraded by endonuclease into 180bp-200bp fragments belonging to hierarchical breaks. Apoptosis is the more common form of cell death.
At present, the research on the core molecule regulation mechanism of the apoptosis signal pathway becomes a key means for treating tumors and becomes an important problem which needs to be solved urgently in the research and development field of tumor drugs.
Disclosure of Invention
Aiming at the difficulty of determining the regulation and control of the core signal molecules of the apoptosis and explaining the change of the core molecules in tumor cells in the prior art, the invention provides a PCR reagent for detecting apoptosis signal pathways and application thereof. The invention concentrates the signal molecules related to cell signal apoptosis on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, discusses the possible target molecules and target ways causing the change of an apoptosis signal channel in the cells and provides the most direct evidence for researching the regulation of the core cell apoptosis signal protein.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a reagent for detecting apoptotic signaling pathways comprising the following primers:
(1) the PCR reaction primers for detecting the ABL1 gene have the following primer sequences:
5'-ATCACGCCAGTCAACAGTCT-3'(SEQ ID NO:1)；
5'-TACACCCTCCCTTCGTATCT-3'(SEQ ID NO:2)；
(2) the primer sequence of the PCR reaction primer for detecting the AIFM1 gene is as follows:
5'-TCCAGCGATGGCATGTTCC-3'(SEQ ID NO:3)；
5'-CCTACTGTTGATAAGCCCACA-3'(SEQ ID NO:4)；
(3) the PCR reaction primers for detecting the AKT1 gene have the following primer sequences:
5'-GTGATCCTGGTGAAGGAGA-3'(SEQ ID NO:5)；
5'-TTAATGTGCCCGTCCTTGT-3'(SEQ ID NO:6)；
(4) the PCR reaction primers for detecting the APAF1 gene have the following primer sequences:
5'-CACACGGTTGGATCAGGAT-3'(SEQ ID NO:7)；
5'-GGAGACGGTCTTTAGCCTCT-3'(SEQ ID NO:8)；
(5) the PCR reaction primer for detecting BAD gene has the following primer sequence:
5'-CCAGAGTTTGAGCCGAGTG-3'(SEQ ID NO:9)；
5'-CCATCCCTTCGTCGTCCT-3'(SEQ ID NO:10)；
(6) the PCR reaction primers for detecting the BAG1 gene have the following primer sequences:
5'-CACACTGATCCTGCCAGAAA-3'(SEQ ID NO:11)；
5'-ACACTCGGCTAGGAATGCC-3'(SEQ ID NO:12)；
(7) the PCR reaction primers for detecting the BAG3 gene have the following primer sequences:
5'-TTCCGGTGATACACGAGCAG-3'(SEQ ID NO:13)；
5'-CTGGTGGGTCTGGTACTC-3'(SEQ ID NO:14)；
(8) the PCR reaction primers for detecting the BAK1 gene have the following primer sequences:
5'-TTTTCCGCAGCTACGTTTTT-3'(SEQ ID NO:15)；
5'-CAGAGGTAAGGTGACCATCTC-3'(SEQ ID NO:16)；
(9) the PCR reaction primer for detecting the BAX gene has the following primer sequence:
5'-GACGAACTGGACAGTAA-3'(SEQ ID NO:17)；
5'-CCTTGAGCACCAGTTT-3'(SEQ ID NO:18)；
(10) the PCR reaction primers for detecting the BCL10 gene have the following primer sequences:
5'-CAGTTGTGAACCTTTTCCAGA-3'(SEQ ID NO:19)；
5'-GGATGCCCTCAGTTTTTCAG-3'(SEQ ID NO:20)；
(11) the PCR reaction primers for detecting the BCL2 gene have the following primer sequences:
5'-AGAGCAGACGGATGGAAAAAGG-3'(SEQ ID NO:21)；
5'-GGCAAAGAAATGCAAGTGAATG-3'(SEQ ID NO:22)；
(12) the PCR reaction primers for detecting the BCL2A1 gene have the following primer sequences:
5'-ACAGGCTGGCTCAGGACTAT-3'(SEQ ID NO:23)；
5'-GCAACATTTTGTAGCACTCTG-3'(SEQ ID NO:24)；
(13) the PCR reaction primers for detecting the BCL2L1 gene have the following primer sequences:
5'-AGCTGGTGGTTGACTTTCTC-3'(SEQ ID NO:25)；
5'-CCATCTCCGATTCAGTCCCT-3'(SEQ ID NO:26)；
(14) the PCR reaction primers for detecting the BCL2L10 gene have the following primer sequences:
5'-CCAGGTTACGGCAGATTCA-3'(SEQ ID NO:27)；
5'-AAGGTCACGAGCGTCACC-3'(SEQ ID NO:28)；
(15) the PCR reaction primers for detecting the BCL2L11 gene have the following primer sequences:
5'-AAGTTCTGAGTGTGACCGAGA-3'(SEQ ID NO:29)；
5'-CTCTGTCTGTAGGGAGGTAGG-3'(SEQ ID NO:30)；
(16) the PCR reaction primers for detecting the BCL2L2 gene have the following primer sequences:
5'-CGGAGTTCACAGCTCTATAC-3'(SEQ ID NO:31)；
5'-AAAGGCCCCTACAGTTACCA-3'(SEQ ID NO:32)；
(17) the PCR reaction primers for detecting the BFAR gene have the following primer sequences:
5'-CAAGACGCCCTATACCATAGA-3'(SEQ ID NO:33)；
5'-GCCTAATGCTTTGACACGTT-3'(SEQ ID NO:34)；
(18) the PCR reaction primer for detecting BID gene has the following primer sequence:
5'-GACTGTGAGGTCAACAACG-3'(SEQ ID NO:35)；
5'-GAAGCCAAACACCAGTAGG-3'(SEQ ID NO:36)；
(19) the PCR reaction primer for detecting the BIK gene has the following primer sequence:
5'-ACCTGGACCCTATGGAGGAC-3'(SEQ ID NO:37)；
5'-CTCAGTCTGGTCGTAGATGA-3'(SEQ ID NO:38)；
(20) the PCR reaction primers for detecting the BIRC2 gene have the following primer sequences:
5'-GCACGATCTTGTCAGATTGG-3'(SEQ ID NO:39)；
5'-GCGGGGAAAGTTGAATATGTA-3'(SEQ ID NO:40)；
(21) the PCR reaction primers for detecting the BIRC3 gene have the following primer sequences:
5'-AGCTACCTCTCAGCCTACTTT-3'(SEQ ID NO:41)；
5'-CACTGTTTTCTGTACCCGGA-3'(SEQ ID NO:42)；
(22) the PCR reaction primers for detecting the BIRC5 gene have the following primer sequences:
5'-GGACCACCGCATCTCTACAT-3'(SEQ ID NO:43)；
5'-AGTCTGGCTCGTTCTCAGTG-3'(SEQ ID NO:44)；
(23) the PCR reaction primers for detecting the BIRC6 gene have the following primer sequences:
5'-GCACAGTTTCCTTGTACGGA-3'(SEQ ID NO:45)；
5'-AGCTTGGGTCTCCTGATAGAA-3'(SEQ ID NO:46)；
(24) the PCR reaction primers for detecting the BNIP2 gene have the following primer sequences:
5'-CCTAGTGATGGCTCTGTATTGT-3'(SEQ ID NO:47)；
5'-CTATTCTCTGACGGTGTGTCT-3'(SEQ ID NO:48)；
(25) the PCR reaction primers for detecting the BNIP3 gene have the following primer sequences:
5'-CAGGGCTCCTGGGTAGAACT-3'(SEQ ID NO:49)；
5'-TACTCCGTCCAGACTCATGC-3'(SEQ ID NO:50)；
(26) the PCR reaction primers for detecting the BNIP3L gene have the following primer sequences:
5'-GTCCAGTAGACCCGAAAACA-3'(SEQ ID NO:51)；
5'-GTGCTCAGTCGCTTTCCAAT-3'(SEQ ID NO:52)；
(27) the PCR reaction primer for detecting the BRAF gene has the following primer sequence:
5'-CCCAAGTCACCACAAAAACC-3'(SEQ ID NO:53)；
5'-GGACTGTAACTCCACACCTT-3'(SEQ ID NO:54)；
(28) the PCR reaction primers for detecting the CASP1 gene have the following primer sequences:
5'-TTCCGCAAGGTTCGATTTTCA-3'(SEQ ID NO:55)；
5'-GCATCTGCGCTCTACCATC-3'(SEQ ID NO:56)；
(29) the PCR reaction primers for detecting the CASP10 gene have the following primer sequences:
5'-CTTCCCAAAACTGAAATGACC-3'(SEQ ID NO:57)；
5'-CTTGATACGACTCGGCTTCC-3'(SEQ ID NO:58)；
(30) the PCR reaction primers for detecting the CASP14 gene have the following primer sequences:
5'-GCTGGAGAATCTCTTCGAGG-3'(SEQ ID NO:59)；
5'-CGTGCAAGGCATCTGTGTAT-3'(SEQ ID NO:60)；
(31) the PCR reaction primers for detecting the CASP2 gene have the following primer sequences:
5'-GCTGTTGTTGAGCGAATTGT-3'(SEQ ID NO:61)；
5'-GCAAGTTGAGGAGTTCCACA-3'(SEQ ID NO:62)；
(32) the PCR reaction primers for detecting the CASP3 gene have the following primer sequences:
5'-ATGGAAGCGAATCAATGGACT-3'(SEQ ID NO:63)；
5'-TGTACCAGACCGAGATGTCA-3'(SEQ ID NO:64)；
(33) the PCR reaction primers for detecting the CASP4 gene have the following primer sequences:
5'-CTGCGGAACTGTGCATGATG-3'(SEQ ID NO:65)；
5'-GTGTGATGAAGATAGAGCCCAT-3'(SEQ ID NO:66)；
(34) the PCR reaction primers for detecting the CASP5 gene have the following primer sequences:
5'-TCAACACCACATAACGTGTCC-3'(SEQ ID NO:67)；
5'-TCAAGGTTGCTCGTTCTATGG-3'(SEQ ID NO:68)；
(35) the PCR reaction primers for detecting the CASP6 gene have the following primer sequences:
5'-ACCAACATAACTGAGGTGGATG-3'(SEQ ID NO:69)；
5'-GGAGGAGCCATATTTTCCCA-3'(SEQ ID NO:70)；
(36) the PCR reaction primers for detecting the CASP7 gene have the following primer sequences:
5'-GGGACCGAGCTTGATGATG-3'(SEQ ID NO:71)；
5'-ACTGGGATCTTGTATCGAGGA-3'(SEQ ID NO:72)；
(37) the PCR reaction primers for detecting the CASP8 gene have the following primer sequences:
5'-TTCTGCCTACAGGTTCCACT-3'(SEQ ID NO:73)；
5'-CTCAATTCTGATCTGCTCACTT-3'(SEQ ID NO:74)；
(38) the PCR reaction primers for detecting the CASP9 gene have the following primer sequences:
5'-TGTCTACGGCACAGATGGAT-3'(SEQ ID NO:75)；
5'-GGACTCGTCTTCAGGGGAA-3'(SEQ ID NO:76)；
(39) the PCR reaction primers for detecting the CD27 gene have the following primer sequences:
5'-GCAGAGCCTTGTCGTTACAG-3'(SEQ ID NO:77)；
5'-CTCCGGTTTTCGGTAATCCT-3'(SEQ ID NO:78)；
(40) the PCR reaction primers for detecting the CD40 gene have the following primer sequences:
5'-CTGAAACGGAATGCCTTCCT-3'(SEQ ID NO:79)；
5'-CTCACTCGTACAGTGCCA-3'(SEQ ID NO:80)；
(41) the PCR reaction primers for detecting the CD40LG gene have the following primer sequences:
5'-CTCCATTTATAGCCAGCCTC-3'(SEQ ID NO:81)；
5'-TTTGGCGGAACTGTGGGT-3'(SEQ ID NO:82)；
(42) the PCR reaction primers for detecting the CD70 gene have the following primer sequences:
5'-ACCCCAGGCTATACTGGCA-3'(SEQ ID NO:83)；
5'-AGGCTGATGCTACGGGAG-3'(SEQ ID NO:84)；
(43) the PCR reaction primer for detecting the CFLAR gene has the following primer sequence:
5'-ACAGAGCTTCTTCGAGACAC-3'(SEQ ID NO:85)；
5'-CTCGGGCATACAGGCAAAT-3'(SEQ ID NO:86)；
(44) the PCR reaction primers for detecting the CIDEA gene have the following primer sequences:
5'-TTGGGAGACAACACGCATTT-3'(SEQ ID NO:87)；
5'-CTCGCTATTCCCGACCTCTT-3'(SEQ ID NO:88)；
(45) the PCR reaction primers for detecting the CIDEB gene have the following primer sequences:
5'-CAAGGACATCGCCCGATT-3'(SEQ ID NO:89)；
5'-TAGAGCCCGTAGAATGTGGC-3'(SEQ ID NO:90)；
(46) the PCR reaction primer for detecting CRDD gene has the following primer sequence:
5'-ATCAGACCGGCAGATTAACC-3'(SEQ ID NO:91)；
5'-TTGGCCTTACAGCGGTAGAT-3'(SEQ ID NO:92)；
(47) the PCR reaction primer for detecting CYCS gene has the following primer sequence:
5'-TTTGGGCGGAAGACAGGTC-3'(SEQ ID NO:93)；
5'-TATTGGCGGCTGTGTAAGAG-3'(SEQ ID NO:94)；
(48) the PCR reaction primers for detecting the DAPK1 gene have the following primer sequences:
5'-CGTGGATGATTACTACGACACC-3'(SEQ ID NO:95)；
5'-GCTTTTCTCACGGCATTTCT-3'(SEQ ID NO:96)；
(49) the primer sequence of the PCR reaction primer for detecting the DFFA gene is as follows:
5'-CCTCTTGTCAAAGCAGGAAG-3'(SEQ ID NO:97)；
5'-GTCCGAGGAGGTCTCTCT-3'(SEQ ID NO:98)；
(50) the primer sequence of the PCR reaction for detecting the DIABLO gene is as follows:
5'-GCGCAGCGTAACTTCATTC-3'(SEQ ID NO:99)；
5'-CAAAGCCAATCGTCACAGTTTT-3'(SEQ ID NO:100)；
(51) the primer sequence of the PCR primer for detecting the FADD gene is as follows:
5'-CTGGCTCGTCAGCTCAAA-3'(SEQ ID NO:101)；
5'-CTGTTGCGTTCTCCTTCTCT-3'(SEQ ID NO:102)；
(52) the primer sequence of the PCR reaction primer for detecting the FAS gene is as follows:
5'-AAGGGATTGGAATTGAGGA-3'(SEQ ID NO:103)；
5'-AGGCCTTCCAAGTTCTGAG-3'(SEQ ID NO:104)；
(53) the primer sequence of the PCR reaction primer for detecting the FASLG gene is as follows:
5'-TGCCTTGGTAGGATTGGGC-3'(SEQ ID NO:105)；
5'-CTGGTAGACTCTCGGAGTTC-3'(SEQ ID NO:106)；
(54) the PCR reaction primers for detecting the GADD45A gene have the following primer sequences:
5'-CCTGATCCAGGCGTTTTG-3'(SEQ ID NO:107)；
5'-ATCCATGTAGCGACTTTCCC-3'(SEQ ID NO:108)；
(55) the PCR reaction primers for detecting the HRK gene have the following primer sequences:
5'-GGTTCCCGTTTTCCAGAGG-3'(SEQ ID NO:109)；
5'-GCAGCTGGATTTCCAAAGG-3'(SEQ ID NO:110)；
(56) the PCR reaction primer for detecting IGF1R gene has the following primer sequence:
5'-TGCTGACCTCTGTTACCTCT-3'(SEQ ID NO:111)；
5'-GCTTATTCCCCACAATGTAGTT-3'(SEQ ID NO:112)；
(57) the primer sequence of the PCR reaction primer for detecting the IL10 gene is as follows:
5'-GACTTTAAGGGTTACCTGGGTTG-3'(SEQ ID NO:113)；
5'-TCACATGCGCCTTGATGTCTG-3'(SEQ ID NO:114)；
(58) the PCR reaction primer for detecting LTA gene has the following primer sequence:
5'-ATGACACCACCTGAACGTCTC-3'(SEQ ID NO:115)；
5'-CTCTCCAGAGCAGTGAGTTCT-3'(SEQ ID NO:116)；
(59) the PCR reaction primers for detecting the LTBR gene have the following primer sequences:
5'-CTCAGCTAAATGTAGCCGCAT-3'(SEQ ID NO:117)；
5'-ATGGTCAGGTAGTTCCAGTGC-3'(SEQ ID NO:118)；
(60) the PCR reaction primers for detecting the MCL1 gene have the following primer sequences:
5'-TGCCTTTGTGGCTAAACACT-3'(SEQ ID NO:119)；
5'-GTCCCGTTTTGTCCTTACGA-3'(SEQ ID NO:120)；
(61) the PCR reaction primer for detecting the NAIP gene has the following primer sequence:
5'-CCATTAGACGATCACACCAGA-3'(SEQ ID NO:121)；
5'-GAGTCACTTCCGCAGAGG-3'(SEQ ID NO:122)；
(62) PCR reaction primers for detecting NFKB1 gene have the following primer sequences:
5'-ACAGAGAGGATTTCGTTTCCG-3'(SEQ ID NO:123)；
5'-TTGACCTGAGGGTAAGACTTCT-3'(SEQ ID NO:124)；
(63) the primer sequence of the PCR reaction primer for detecting the NOD1 gene is as follows:
5'-CTGAAAAGCAATCGGGAACTT-3'(SEQ ID NO:125)；
5'-ACACACAATCTCCGCATCTT-3'(SEQ ID NO:126)；
(64) the primer sequence of the PCR reaction primer for detecting the NOL3 gene is as follows:
5'-ACCGCAGCTATGACCCTC-3'(SEQ ID NO:127)；
5'-TCCGGTTCAGCCTCTTTAGA-3'(SEQ ID NO:128)；
(65) the PCR reaction primer for detecting the PYCARD gene has the following primer sequence:
5'-GGATGCTCTGTACGGGAAG-3'(SEQ ID NO:129)；
5'-CAGGCTGGTGTGAAACTGAA-3'(SEQ ID NO:130)；
(66) the PCR reaction primers for detecting the RIPK2 gene have the following primer sequences:
5'-TGCCATTCACCTATGTGACA-3'(SEQ ID NO:131)；
5'-GACAGTGATGCAGCTTCATAAA-3'(SEQ ID NO:132)；
(67) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'-CTCTCTCTAATCAGCCCTCTG-3'(SEQ ID NO:133)；
5'-AGGACCTGGGAGTAGATGAG-3'(SEQ ID NO:134)；
(68) the PCR reaction primers for detecting the TNFRSF10A gene have the following primer sequences:
5'-CGGGGAGGATTGAACCAC-3'(SEQ ID NO:135)；
5'-GACGACAAACTTGAAGGTCTT-3'(SEQ ID NO:136)；
(69) the PCR reaction primers for detecting the TNFRSF10B gene have the following primer sequences:
5'-TGGAACAACGGGGACAGAAC-3'(SEQ ID NO:137)；
5'-TGCTGGGGAGCTAGGTCT-3'(SEQ ID NO:138)；
(70) the PCR reaction primers for detecting the TNFRSF11B gene have the following primer sequences:
5'-ACAAATTGCAGTGTCTTTGGTC-3'(SEQ ID NO:139)；
5'-CTGCGTTTACTTTGGTGCCA-3'(SEQ ID NO:140)；
(71) the PCR reaction primers for detecting the TNFRSF1A gene have the following primer sequences:
5'-CGCTCGTGTTTCTGGACA-3'(SEQ ID NO:141)；
5'-GTATAGACACTCGTCACTGGTG-3'(SEQ ID NO:142)；
(72) the PCR reaction primers for detecting the TNFRSF1B gene have the following primer sequences:
5'-TCATCCACGGATATTTGCAGG-3'(SEQ ID NO:143)；
5'-CTGGGGTAAGTGTACTGCC-3'(SEQ ID NO:144)；
(73) the PCR reaction primers for detecting the TNFRSF21 gene have the following primer sequences:
5'-TGACTGACCGAGAATGCACT-3'(SEQ ID NO:145)；
5'-TCATCACACTAGAAGGCACATC-3'(SEQ ID NO:146)；
(74) the PCR reaction primers for detecting the TNFRSF25 gene have the following primer sequences:
5'-TGGCAGCGGGCACATTCAGAATT-3'(SEQ ID NO:147)；
5'-CTTGCACGGAGCCCTGCGGCAA-3'(SEQ ID NO:148)；
(75) the PCR reaction primers for detecting the TNFRSF9 gene have the following primer sequences:
5'-TGGATGGAAAGTCTGTGCTTG-3'(SEQ ID NO:149)；
5'-GGAGATGATCTGCGGAGAGT-3'(SEQ ID NO:150)；
(76) the PCR reaction primers for detecting the TNFSF10 gene have the following primer sequences:
5'-CCACCCTGACCTACACATAC-3'(SEQ ID NO:151)；
5'-TTACCCACCAACTGGACGG-3'(SEQ ID NO:152)；
(77) the PCR reaction primers for detecting the TNFSF8 gene have the following primer sequences:
5'-GAGGACGGACTCCATTCCC-3'(SEQ ID NO:153)；
5'-GTAGGCCCATGACTTCTTGAA-3'(SEQ ID NO:154)；
(78) the primer sequence of the PCR reaction primer for detecting the TP53 gene is as follows:
5'-AGCACATGACGGAGGTTGT-3'(SEQ ID NO:155)；
5'-CATCCAAATACTCCACACGC-3'(SEQ ID NO:156)；
(79) the primer sequence of the PCR reaction primer for detecting the TP53BP2 gene is as follows:
5'-AAGTGCGTCCGTTCTCAATG-3'(SEQ ID NO:157)；
5'-GTTCTTCCTCAGAGTACCAAAG-3'(SEQ ID NO:158)；
(80) the primer sequence of the PCR reaction primer for detecting the TP73 gene is as follows:
5'-ACGAGGACACGTACTACCTT-3'(SEQ ID NO:159)；
5'-TGCCGATAGGAGTCCACCA-3'(SEQ ID NO:160)；
(81) the PCR reaction primer for detecting the TRADD gene has the following primer sequence:
5'-ATTCTGCCTCAGGTACTT-3'(SEQ ID NO:161)；
5'-CAGGACACCAAAGATCAA-3'(SEQ ID NO:162)；
(82) the primer sequence of the PCR reaction primer for detecting the TRAF2 gene is as follows:
5'-CCCTGGAGTTGCTACAGC-3'(SEQ ID NO:163)；
5'-GGCGGAGCACAGGTACTT-3'(SEQ ID NO:164)；
(83) the primer sequence of the PCR reaction primer for detecting the TRAF3 gene is as follows:
5'-AGACTAACCCGCCGCTAAAG-3'(SEQ ID NO:165)；
5'-ATGCTCTCTTGACACGCTGT-3'(SEQ ID NO:166)；
(84) the primer sequence of the PCR reaction primer for detecting the XIAP gene is as follows:
5'-CCGTGCGGTGCTTTAGTT-3'(SEQ ID NO:167)；
5'-GCGTGGCACTATTTTCAAGATA-3'(SEQ ID NO:168)；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'-AGACCTTCAACACCCCAGC-3'(SEQ ID NO:169)；
5'-TGTCACGCACGATTTCCC-3'(SEQ ID NO:170)；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'-CCAGAAGTACAGAAAACATGG-3'(SEQ ID NO:171)；
5'-CAAGGAGCCTATGACAGTGAA-3'(SEQ ID NO:172)；
(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'-CTCTCTGCTCCTCCTGTTC-3'(SEQ ID NO:173)；
5'-CGACCAAATCCGTTGACTC-3'(SEQ ID NO:174)；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'-CTGGCGTCGTGATTAGTGAT-3'(SEQ ID NO:175)；
5'-GACGTTCAGTCCTGTCCATAA-3'(SEQ ID NO:176)；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'-CATTCTATCATCAACGGGTACAA-3'(SEQ ID NO:177)；
5'-CAGCAAGTGGGAAGGTGTAATC-3'(SEQ ID NO:178)。
the invention also provides application of the PCR reagent in preparation of a kit for detecting an apoptosis signal pathway.
Further: the cells comprise cancer cells and normal cells, and the cancer cells are oral squamous carcinoma cells, breast cancer cells, lung cancer cells and liver cancer cells.
Further: the kit uses a real-time fluorescent quantitative PCR detection method for detection.
Further: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1 mul-0.2 mul of upstream primer, 0.1 mul-0.2 mul of downstream primer, 25ng-100ng of cDNA template, and sterile distilled water to make up to 20 mul.
Further: the Taq polymerase is TaKaRa
Figure BDA0002580759510000081
Premix Ex Taq polymerase.
Further: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
Compared with the prior art, the invention has the advantages and positive effects that: the invention provides a PCR reagent for detecting the change of apoptosis signal channel core molecules, which can rapidly detect genes related to apoptosis at the transcription level by real-time fluorescence quantitative apoptosis, and analyze and research the core molecules and action mechanism of tumor apoptosis signal channel on the basis, thereby rapidly and accurately finding out the apoptosis regulating signal channel of tumor related drugs, and providing a powerful tool for anti-cancer drug screening, mechanism discussion of new targeted drugs and the like.
The invention determines the gene related to the apoptosis signal channel through a large number of creative experiments, the invention focuses on the combination of primers, and experiments prove that the situations of primer dimer and non-specific amplification can be avoided only through proper primer combination during PCR amplification, thereby ensuring the effective amplification of all target genes. The primer dimer often appears in the combination of any primer during the specific PCR, which affects the failure of the target gene amplification, or the non-specific amplification, which greatly affects the effectiveness of the PCR result. Under the same experimental condition, PCR detection of 89 genes (84 of which are detection genes and 5 of which are reference genes) is carried out simultaneously, so that not only is primer design required, but also the optimal amplification condition is required to be well grasped, which is a technical difficulty. The most central is primer design, the invention obtains the best amplification condition through multiple PCR and adjustment, and ensures that the amplification efficiency of each gene is very high without nonspecific amplification.
The invention also provides a PCR method for detection, the experimental system can be carried out on any real-time fluorescent quantitative PCR instrument, and the PCR reaction instrument is LightCycler480 of Roche. The 89 pairs of primers were placed in a 96-well plate and subjected to real-time fluorescent quantitative PCR detection. The experimental system can be carried out on any real-time fluorescence quantitative PCR instrument, can obtain the change of the apoptosis signal channel core molecule through one-time reaction, has simple and convenient experimental operation, low cost and good result repeatability, is beneficial to reducing unnecessary downstream detection, and is an important means for researching the action mechanism of the tumor-related medicament.
Other features and advantages of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 shows the total RNA extracted from the epithelial cell line HaCaT and the oral squamous cell carcinoma cell line SCC-25 according to the present invention, wherein 1: HaCaT cells; 2: SCC-25 cells.
FIG. 2 shows the real-time fluorescent quantitative PCR detection of apoptosis signaling pathway gene expression.
FIG. 3 shows a lysis curve analysis of apoptosis signaling pathway gene expression.
FIG. 4 shows the difference in apoptosis signaling pathway gene expression in HaCaT and the oral squamous carcinoma cell line SCC-25.
FIG. 5 shows the results of a core apoptotic molecule expression screen.
FIG. 6 shows flow cytometry detection of apoptosis of SCC-25 cells.
FIG. 7 shows that Hoechst staining detects apoptosis of SCC-25 cells.
FIG. 8 shows the expression of Bcl-2 and c-Myc proteins under different treatment conditions.
FIG. 9 shows the regulatory relationship between apoptosis-related molecules analyzed using STRING.
FIG. 10 shows the results of a search for the effect of CIDEA and RIPK2 in tumors.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
Example 1
The invention is illustrated by oral squamous cell carcinoma cells, but the invention is not limited to oral squamous cell carcinoma, and the detection reagent of the invention can be applied to common cancers such as breast cancer, lung cancer, liver cancer and the like.
The invention is characterized by culturing two cell lines: human epithelial cell HaCaT and human oral squamous carcinoma cell line SCC-25, and then detecting the change of the apoptosis molecules of the core cells in the two cell lines by using an apoptosis signal path detection reagent.
The human epithelial cell HaCaT and the human oral squamous cell line SCC-25 used in the present invention were purchased from ATCC company, DMEM medium and fetal bovine serum from Gibco company, RNAiso for total RNA extraction, reverse transcription kit,
Figure BDA0002580759510000091
the Premix Ex Taq kit was purchased from TaKaRa.
The reagent for detecting the intracellular apoptosis signal pathway comprises the following specific steps:
first, culture and passage of human epithelial cell HaCaT and human oral squamous carcinoma cell line SCC-25
Taking a human oral squamous carcinoma cell line SCC-25 as an example, 1mL of 0.2% pancreatin solution is added into a 60mm cell culture plate, the pancreatin is gently shaken to be fully contacted with cells, the cell culture plate is incubated in a carbon dioxide incubator at 37 ℃ for 2min, after most of cells are observed to float under a mirror, 4mL of DMEM medium containing 10% FBS is added, the DMEM medium is evenly blown and added into a 15mL centrifugal tube, the 15 g centrifugal tube is centrifuged for 6min, the supernatant is discarded, a corresponding proper volume of culture solution is added according to the cell types, and the cell is blown and beaten repeatedly by a 10mL pipette for a plurality of times to free the cells. In this process, air bubbles are prevented from being generated, so as not to influence the cell count. Taking 20 mu L of cell suspension, dripping into a cell counting plate, and counting the total number of cells in four big grids at four corners according to a cell counting method. When counting, only cells with intact cell morphology are counted, and the number of stacked cells is counted as 1 cell. The calculation formula is as follows:
cell concentration (one/mL) ═ 4 big grid cell count × 104X dilution factor/4.
Diluting to 5 × 10 with corresponding culture solution according to cell type6Adding 2mL of cell dilution into 60mm cell culture dish, placing at 37 deg.C and 5% CO2The cells are cultured in a cell culture box, the cells are attached to the wall after 0.5h-1h generally, the cells begin to grow after a plurality of hours, and the cells become monolayer cells after 24h-48 h.
Second, extraction of Total RNA
1) Taking CO2After sucking out the culture medium from a disk of cells in a 60mm plate in logarithmic phase in an incubator, washing the disk of cells for 2 times by using sterile 1 XPBS, adding 1mL of RNAasso on the surfaces of the cells, repeatedly sucking the cells uniformly by using a gun head, and transferring the cells into a sterile tube.
2) Incubate at room temperature for 5min to solubilize the nucleoprotein. Add 0.2mL phenol/chloroform (1:1), shake vigorously for 15sec, incubate at room temperature for 2-3 min.
3) Cells were stratified by centrifugation at 12000g for 15min at 4 ℃.
4) Transferring the upper layer liquid to a centrifuge tube without RNase, adding 0.5mL isopropanol, reversing and mixing uniformly for 3 times, and incubating for 10min at room temperature.
5) Centrifugation at 12000g for 10min at 4 ℃ resulted in the formation of a bottom pellet of RNA.
6) The supernatant was discarded and the pellet was washed with 1mL of RNase-free 75% ethanol. Centrifuge at 7000g for 5min at 4 ℃.
8) The RNA was air dried for about 5-10 min. It should not be dried too much.
9) RNA was dissolved in 30-50. mu.L DEPC water.
The results are shown in FIG. 1, and it can be seen from the electrophoresis in the figure that Lane 1 is HaCaT, Lane 2 is human oral squamous carcinoma cell line SCC-25 cell, the total RNA band is complete, and it is suitable for downstream reverse transcription and other experiments.
Synthesis of cDNA
1) The following reaction mixture was added to a pre-cooled tube:
1-5 mug of total RNA;
oligo(dT)(0.5μg/μl) 1μL；
diluting to 12 μ L with RNase-free water;
2) mixing, and centrifuging for 3-5 sec.
3) Acting at 70 deg.C for 5min, ice-cooling for 30sec, and centrifuging for 3-5 sec.
4) The tube was ice-cooled and the following components were added:
5×Reaction buffer 4μL
Rnase Inhibiter(20U/μL) 1μL
dNTP mix(10mM)2μL
5) mix gently and centrifuge for 3-5 sec.
6) After being bathed at 37 ℃ for 5min, 1. mu.L of M-MuLV Reverse Transcriptase (20U/. mu.L) was added and mixed well.
7) Acting at 37 deg.C for 60 min. The reaction was terminated by incubation at 70 ℃ for 10min and downstream testing was performed on ice.
Four, real-time fluorescent quantitative PCR
Taking the synthesized cDNA as a template, and carrying out real-time fluorescence quantitative PCR reaction according to the following system:
Figure BDA0002580759510000101
premix Ex Taq 10. mu.L; upstream primer 0.1-0.2 μmol; downstream primer 0.1-0.2 μmol; 25-100ng of cDNA template; DW made up the volume to 20. mu.L.
The following program was set up on the LightCycler480 instrument from roche: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
The upstream and downstream primers in the PCR reaction are respectively the PCR reagents (including 84 primers of the detection genes and 5 primers of the reference genes) described in the invention, and the sequences are shown as SEQ ID NO:1-SEQ ID NO: 178.
The primers are placed on a 96-well plate for PCR reaction, wherein each pair of primers is placed in any one well, and 89 pairs of primers are placed in 89 wells in total. FIG. 2 shows the real-time fluorescence quantitative PCR amplification result of cells, and it can be seen from the amplification curve that the target gene is well amplified and is suitable for downstream Ct value analysis.
Fifth, data analysis
And calculating the expression difference of the apoptosis gene of the core cell by using the Ct value of each gene. FIG. 3 is a dissolution curve analysis after real-time fluorescence quantitative PCR of cells, and from the dissolution curve, the peak shape of the target gene is single, the amplification product is the target gene, and the phenomena of primer dimer, non-specific amplification and the like do not exist, which indicates that the primer and the PCR system are good and are suitable for downstream experiments.
FIG. 4 shows the results of differential expression analysis after real-time fluorescence quantitative PCR of cells, from which apoptosis signaling pathway molecules closely related to the development and progression of oral squamous cell carcinoma can be screened. The screened NAIP, CIDEA, DAPK1 and the like are closely related to the apoptosis of oral squamous cell carcinoma, the genes show that the genes are negatively related to the expression of SCC-25 cells on the map, while CASP10, TNFRSF9, RIPK2, TNF, TNFRSF25 and the like are closely related to the anti-apoptosis of the oral squamous cell carcinoma, and show that the genes are positively related to the expression of the SCC-25 cells on the map.
FIG. 5 core apoptotic molecule expression screening results. The screened core apoptosis molecules are detected again by adopting fluorescence quantitative PCR, and the results are highly consistent with the screened results in the figure 4, namely NAIP, CIDEA, DAPK1 and the like are closely related to the apoptosis of oral squamous cell carcinoma, CASP10, TNFRSF9, RIPK2, TNF, TNFRSF25 and the like are closely related to the anti-apoptosis of the oral squamous cell carcinoma, and the results strongly prove the rapidness and the effectiveness of the apoptosis signal path kit in screening apoptosis signal paths, thereby providing an effective research direction for related functional experiments of downstream apoptosis and the like.
Sixthly, detecting the influence of CIDEA and RIPK2 on the apoptosis of oral squamous cell carcinoma by adopting a flow cytometer
In normal cells, Phosphatidylserine (PS) on the cell membrane is located on the cytosolic side. Mid-apoptotic, PS eversion. Annexin V is Ca2+Dependent on phospholipid binding proteins with high affinity for PS, eversion of PS can be detected with fluorescently labeled Annexin V. At the end of apoptosis, the cell membrane structure is damaged, and nucleic acid dyes (such as PI, 7-AAD and the like) which are originally impermeable to the membrane can enter the cell, thereby generating signals. The signals of Annexin V and the membrane-impermeable nucleic acid dye are respectively detected, and the proportion of cells in the middle and end apoptosis stages can be quantified. Therefore, the screened core apoptosis molecules are detected by a flow cytometer, taking CIDEA and RIPK2 as examples.
The total of the two groups is divided into four groups, namely a pcDNA Control group (OE-Control), an overexpression CIDEA group (OE-CIDEA), an sh-RNA Control group and an sh-RIPK2 group.
Collecting cells: taking a proper amount of SCC-25 cells in the logarithmic phase of growth to inoculate in a 6-well plate, treating for a corresponding time under corresponding conditions (such as the above various treatment groups), collecting the cells, and taking attention to combine the supernatant and the digested cells (note: directly centrifuging the suspension cells);
and (3) cleaning cells: washing 2 times with pre-cooled PBS;
grouping: each experiment is divided into a non-dyeing group, a single-dyeing Annexin V group, a single-dyeing PI group and a PI and Annexin V double-dyeing group, and the treatment group is subjected to double-dyeing from low to high;
dyeing: the 4 Xbinding buffer was diluted to 1 Xbuffer with PBS, the residual PBS in the centrifuge tube was aspirated, 100. mu.L of 1 Xbinding buffer was added to each tube, the cells were blown with a pipette to resuspend them thoroughly, and the dye was added in the dark. Adding Annexin V or PI 5 muL into a single-dyeing group, adding Annexin V and PI 5 muL into a double-dyeing group, and gently mixing by using a pipette gun;
and (3) computer detection: after incubation for 15 minutes at room temperature in the dark, 300. mu.L of binding buffer was added and mixed well, and the cell suspension was transferred to a 5mL flow tube in the dark for 1h on-machine detection on a FACSCalibur flow cytometer.
As shown in FIG. 6, compared with the pcDNA Control group (OE-Control), the over-expression CIDEA group (OE-CIDEA) significantly improved the apoptosis of SCC-25; meanwhile, compared with the sh-RNA control group, the apoptosis number of the group with knockdown expression of sh-RIPK2 is obviously higher than that of the control group.
Seventhly, detecting the influence of CIDEA and RIPK2 on oral squamous cell carcinoma apoptosis by hoechst
Since one of the morphological features of apoptotic cells is chromatin condensation and the disintegration of nuclear structures in the late stages of apoptosis, the observation of nuclear morphology is also a more direct indicator. Compared with other morphological indexes, the nuclear morphology can be observed only by using fluorescent dye and matching with a common fluorescence microscope.
The nuclear morphology staining adopts Hoechst series fluorescent dye. For the living cells SCC-25 in the culture vessel, a proper amount of Hoechst 33342 living cell staining solution (100X) was added dropwise to the culture solution to a final concentration of 1X, and the mixture was gently mixed. For cells cultured in twelve-well plates, 1ml of culture medium was added per well, and 10. mu.l of Hoechst 33342 viable cell staining solution (100X) was added per well. Serum, phenol red and the like in the culture solution do not interfere with the dyeing.
Incubate in cell incubator for 10 min. Absorbing the culture solution containing the dye, washing with the culture solution or PBS for 2-3 times, and observing under a fluorescence microscope. When the cell is apoptotic, the cell nucleus of the apoptotic cell is densely and densely stained or is densely and densely stained in a broken block shape. The results are shown in FIG. 7: compared with a pcDNA Control group (OE-Control), the over-expression CIDEA group (OE-CIDEA) remarkably improves the apoptosis of SCC-25; meanwhile, compared with the sh-RNA control group, the apoptosis number of the group with knockdown expression of sh-RIPK2 is obviously higher than that of the control group.
Eighthly, detecting A, B influence on oral squamous cell carcinoma apoptosis by Western blot
1. Collecting protein samples
Adherent cells were lysed using RIPA lysate as required for the experiment, and protease inhibitors, phosphatase inhibitors or deacetylase inhibitor cocktail were additionally added to prevent degradation of proteins.
After the protein samples are collected, the protein concentration of each protein sample needs to be determined in order to ensure that the loading amount of each protein sample is consistent. Protein concentration was detected using the BCA protein concentration assay kit.
2. Electrophoresis
Preparing polyacrylamide gel, 12% of separation gel and 5% of lamination gel.
100ml of Bio-Rad 10 Xelectrophoresis buffer was dissolved in 900ml of distilled water to prepare an lL electrophoresis buffer, which was prepared as it is.
Prepared as lx buffer (19 × 100ml of stock buffer solution of transmembrane buffer +200ml of methanol (analytically pure) +700ml of deionized water); 100ml 10xTBS buffer from Biochemical company was dissolved in 900ml distilled water to prepare 1L l XTBS buffer, and l ml Tween-20 was added to prepare l XTBST buffer.
0.5ml of skimmed milk and 9.5ml of TBST solution are prepared into a confining liquid containing 5 percent of skimmed milk, and the confining liquid is prepared for use.
0.5ml of skimmed milk and 9.5ml of TBST are prepared into an antibody diluent containing 5% of skimmed milk, wherein the primary antibody diluent contains 0.002g of sodium azide, and the secondary antibody diluent does not contain, so that the antibody diluent is prepared for use.
And putting the prefabricated gel into an electrophoresis device, immersing the electrophoresis device in lx electrophoresis liquid, and checking to determine that no liquid leakage exists.
The power supply is switched on, and the electrophoresis is carried out for 15min at a constant voltage of 80V.
Add 20. mu.g of protein to the loading well and electrophoresis marker to the very edge of the lane.
The power is switched on, and the electrophoresis is carried out for 30min at a constant voltage of 80V and then for 1h at 120V.
And finishing electrophoresis when the lowest color band on the prefabricated glue is 1-2cm away from the lower edge of the glue, and preparing to transfer the film.
3. Rotary film
A PVDF membrane of the same size as the gel was cut out and soaked in anhydrous methanol for 10 seconds.
And sealing the rotary template and then placing the rotary template into a rotary film groove. Adding about 1000ml of pre-cooled membrane transferring liquid with 4 ℃ into the membrane transferring groove, and putting an ice bag into the membrane transferring groove to maintain the low temperature of the whole process.
The electrophoresis tank was placed in ice cubes. And (5) rotating the membrane for 90min at a constant current of 0.2A.
4. Sealing of
Immersing PVDF membrane in 5% skimmed milk, and shaking for 90min at room temperature.
5. Hybridization of
The blocked PVDF membrane was washed with PBST and immersed in primary antibody overnight in a shaker at 4 ℃ for 30min the next day at room temperature. PBST was washed 3 times for 15min each. The secondary antibody was immersed for 1h and washed 3 times with PBST, 15min each time.
6. Development
According to the ECL instruction, the solution A and the solution B are added into a 15ml centrifuge tube according to the proportion of 1:1, mixed by turning upside down for several times, and kept standing for 5min in a dark place. The ECL mixed solution is uniformly dripped on the membrane (preferably 1000 mul of mixed solution per membrane), and the membrane is sealed by preservative film and fixed in a dark box. The film was quickly covered over the ECL film in the dark, the cassette was closed, and the exposure was done according to the intensity of the fluorescence seen (l s-30 min).
Taking out the film, immediately and completely immersing the film into the developing solution for l min, rinsing the film for 30s with clear water, immersing the film into the fixing solution, completely fixing the film after about 30s, washing the film with clear water, drying the film, calibrating the size of the molecular weight of the strip according to the position of a Marker, and analyzing and scanning the film.
Studies have shown that c-Myc and Bcl-2 play an important role in apoptosis of tumor cells, so Western blot is adopted to detect the expression of the c-Myc and the Bcl-2 under different conditions, and the result is shown in figure 8, and after CIDEA is over-expressed, the expression of the c-Myc and the Bcl-2 is obviously reduced. After downregulation of RIPK2 expression, c-Myc and Bcl-2 expression was significantly reduced. The result further proves that the apoptosis-related protein obtained by screening the patent has obvious apoptosis regulation effect and is suitable for screening the apoptosis core protein.
Ninth, adopting STRING to detect the mutual regulation and control relationship of core apoptosis protein
The regulation and control relationship of the core apoptosis protein is detected by bioinformatics, as shown in figure 9, the invention adopts an online STRING website to detect the apoptosis core protein (https:// STRING-db. org /), and the results show that TNFRSF25, TNF, RIPK2, TNFRSF9, CASP10, DAPK1, CIDEA and NAIP obtained by screening the apoptosis signal channel are closely regulated and controlled in a signal system and have forward or (and) reverse regulation and control relationship with each other, so the molecules are closely related to apoptosis.
Through a series of experimental results, the genes closely related to the apoptosis signal pathway of the oral squamous cell carcinoma cell are finally proved to be TNFRSF25, TNF, RIPK2, TNFRSF9, CASP10, DAPK1, CIDEA and NAIP, and the reliability and the effectiveness of the kit and the detection method are fully proved.
Analysis of core apoptosis factor
Apoptosis is a fundamental biological phenomenon of cells that plays a necessary role in the biological elimination of unwanted or abnormal cells by multicellular organisms, particularly in tumor cells: induction of apoptosis in tumor cells is an important approach to the treatment of tumors. Therefore, the screening of the tumor apoptosis (or anti-apoptosis factors) of the core has important practical significance. By taking the CIDEA and RIPK2 screened by the invention as examples, the research mechanism of the factor in the tumor is discussed. First, taking CIDEA as an example, searching the document related to the factor on the website of coremine (http:// www.coremine.com/medical/# search) found that CIDEA is closely related to tumor apoptosis, and the degree of the relationship is ranked at the head of the tumor regulation process (the result is shown in 10A). Secondly, the website of coremine (http:// www.coremine.com/medical/# search) is searched again by RIPK2, the result shows that RIPK2 is also closely related to the apoptosis pathway (the result is shown as 10B), and the result proves the effectiveness of the apoptosis signal pathway detection kit again.
The invention concentrates the molecules closely related to the apoptosis signal path on a flat plate, reacts the survival state of the cell by performing a real-time fluorescent quantitative PCR reaction, compares the survival state with the normal HaCaT cell and oral squamous cell carcinoma, discusses the possible path of the apoptosis signal path in the tumor/normal cell, and provides the most direct evidence for researching the regulation and control of key protein; the invention can quickly and accurately find the apoptosis signal channel related molecules from the transcription level, and provides a powerful tool for screening anti-cancer drugs, discussing the mechanism of new targeted drugs and the like.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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<210>35
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gactgtgagg tcaacaacg 19
<210>36
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
gaagccaaac accagtagg 19
<210>37
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
acctggaccc tatggaggac 20
<210>38
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
ctcagtctgg tcgtagatga 20
<210>39
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
gcacgatctt gtcagattgg 20
<210>40
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
gcggggaaag ttgaatatgt a 21
<210>41
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
agctacctct cagcctactt t 21
<210>42
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
cactgttttc tgtacccgga 20
<210>43
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
ggaccaccgc atctctacat 20
<210>44
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
agtctggctc gttctcagtg 20
<210>45
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gcacagtttc cttgtacgga 20
<210>46
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
agcttgggtc tcctgataga a 21
<210>47
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
cctagtgatg gctctgtatt gt 22
<210>48
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
ctattctctg acggtgtgtc t 21
<210>49
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
cagggctcct gggtagaact 20
<210>50
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
tactccgtcc agactcatgc 20
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gtccagtaga cccgaaaaca 20
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
gtgctcagtc gctttccaat 20
<210>53
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
cccaagtcac cacaaaaacc 20
<210>54
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
ggactgtaac tccacacctt 20
<210>55
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
ttccgcaagg ttcgattttc a 21
<210>56
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
gcatctgcgc tctaccatc 19
<210>57
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
cttcccaaaa ctgaaatgac c 21
<210>58
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
cttgatacga ctcggcttcc 20
<210>59
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
gctggagaat ctcttcgagg 20
<210>60
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
cgtgcaaggc atctgtgtat 20
<210>61
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
gctgttgttg agcgaattgt 20
<210>62
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
gcaagttgag gagttccaca 20
<210>63
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
atggaagcga atcaatggac t 21
<210>64
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
tgtaccagac cgagatgtca 20
<210>65
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
ctgcggaact gtgcatgatg 20
<210>66
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
gtgtgatgaa gatagagccc at 22
<210>67
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
tcaacaccac ataacgtgtc c 21
<210>68
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
tcaaggttgc tcgttctatg g 21
<210>69
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
accaacataa ctgaggtgga tg 22
<210>70
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
ggaggagcca tattttccca 20
<210>71
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
gggaccgagc ttgatgatg 19
<210>72
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
actgggatct tgtatcgagg a 21
<210>73
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
ttctgcctac aggttccact20
<210>74
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
ctcaattctg atctgctcac tt 22
<210>75
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
tgtctacggc acagatggat 20
<210>76
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
ggactcgtct tcaggggaa 19
<210>77
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
gcagagcctt gtcgttacag 20
<210>78
<211>20
<212>DNA
<213> Artificial sequence (artificacial sequence)
<400>78
ctccggtttt cggtaatcct 20
<210>79
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>79
ctgaaacgga atgccttcct 20
<210>80
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>80
ctcactcgta cagtgcca 18
<210>81
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>81
ctccatttat agccagcctc 20
<210>82
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>82
tttggcggaa ctgtgggt 18
<210>83
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>83
accccaggct atactggca 19
<210>84
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>84
aggctgatgc tacgggag 18
<210>85
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>85
acagagcttc ttcgagacac 20
<210>86
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>86
ctcgggcata caggcaaat 19
<210>87
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>87
ttgggagaca acacgcattt20
<210>88
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>88
ctcgctattc ccgacctctt 20
<210>89
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>89
caaggacatc gcccgatt 18
<210>90
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
tagagcccgt agaatgtggc 20
<210>91
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
atcagaccgg cagattaacc 20
<210>92
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
ttggccttac agcggtagat 20
<210>93
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
tttgggcgga agacaggtc 19
<210>94
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
tattggcggc tgtgtaagag 20
<210>95
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>95
cgtggatgat tactacgaca cc 22
<210>96
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>96
gcttttctca cggcatttct 20
<210>97
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>97
cctcttgtca aagcaggaag 20
<210>98
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>98
gtccgaggag gtctctct 18
<210>99
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>99
gcgcagcgta acttcattc 19
<210>100
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>100
caaagccaat cgtcacagtt tt 22
<210>101
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>101
ctggctcgtc agctcaaa18
<210>102
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>102
ctgttgcgtt ctccttctct 20
<210>103
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>103
aagggattgg aattgagga 19
<210>104
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>104
aggccttcca agttctgag 19
<210>105
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>105
tgccttggta ggattgggc 19
<210>106
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>106
ctggtagact ctcggagttc 20
<210>107
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>107
cctgatccag gcgttttg 18
<210>108
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>108
atccatgtag cgactttccc 20
<210>109
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>109
ggttcccgtt ttccagagg 19
<210>110
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>110
gcagctggat ttccaaagg 19
<210>111
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>111
tgctgacctc tgttacctct 20
<210>112
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>112
gcttattccc cacaatgtag tt 22
<210>113
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>113
gactttaagg gttacctggg ttg 23
<210>114
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>114
tcacatgcgc cttgatgtct g 21
<210>115
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>115
atgacaccac ctgaacgtct c21
<210>116
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>116
ctctccagag cagtgagttc t 21
<210>117
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>117
ctcagctaaa tgtagccgca t 21
<210>118
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>118
atggtcaggt agttccagtg c 21
<210>119
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>119
tgcctttgtg gctaaacact 20
<210>120
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>120
gtcccgtttt gtccttacga 20
<210>121
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>121
ccattagacg atcacaccag a 21
<210>122
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>122
gagtcacttc cgcagagg 18
<210>123
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>123
acagagagga tttcgtttcc g 21
<210>124
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>124
ttgacctgag ggtaagactt ct 22
<210>125
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>125
ctgaaaagca atcgggaact t 21
<210>126
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>126
acacacaatc tccgcatctt 20
<210>127
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>127
accgcagcta tgaccctc 18
<210>128
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>128
tccggttcag cctctttaga 20
<210>129
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>129
ggatgctctg tacgggaag 19
<210>130
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>130
caggctggtg tgaaactgaa 20
<210>131
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>131
tgccattcac ctatgtgaca 20
<210>132
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>132
gacagtgatg cagcttcata aa 22
<210>133
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>133
ctctctctaa tcagccctct g 21
<210>134
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>134
aggacctggg agtagatgag 20
<210>135
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>135
cggggaggat tgaaccac 18
<210>136
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>136
gacgacaaac ttgaaggtct t 21
<210>137
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>137
tggaacaacg gggacagaac 20
<210>138
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>138
tgctggggag ctaggtct 18
<210>139
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>139
acaaattgca gtgtctttgg tc 22
<210>140
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>140
ctgcgtttac tttggtgcca 20
<210>141
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>141
cgctcgtgtt tctggaca 18
<210>142
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>142
gtatagacac tcgtcactgg tg 22
<210>143
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>143
tcatccacgg atatttgcag g 21
<210>144
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>144
ctggggtaag tgtactgcc 19
<210>145
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>145
tgactgaccg agaatgcact 20
<210>146
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>146
tcatcacact agaaggcaca tc 22
<210>147
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>147
tggcagcggg cacattcaga att 23
<210>148
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>148
cttgcacgga gccctgcggc aa 22
<210>149
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>149
tggatggaaa gtctgtgctt g 21
<210>150
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>150
ggagatgatc tgcggagagt 20
<210>151
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>151
ccaccctgac ctacacatac 20
<210>152
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>152
ttacccacca actggacgg 19
<210>153
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>153
gaggacggac tccattccc 19
<210>154
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>154
gtaggcccat gacttcttga a 21
<210>155
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>155
agcacatgac ggaggttgt 19
<210>156
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>156
catccaaata ctccacacgc 20
<210>157
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>157
aagtgcgtcc gttctcaatg 20
<210>158
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>158
gttcttcctc agagtaccaa ag 22
<210>159
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>159
acgaggacac gtactacctt 20
<210>160
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>160
tgccgatagg agtccacca 19
<210>161
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>161
attctgcctc aggtactt 18
<210>162
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>162
caggacacca aagatcaa 18
<210>163
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>163
ccctggagtt gctacagc 18
<210>164
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>164
ggcggagcac aggtactt 18
<210>165
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>165
agactaaccc gccgctaaag 20
<210>166
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>166
atgctctctt gacacgctgt 20
<210>167
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>167
ccgtgcggtg ctttagtt 18
<210>168
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>168
gcgtggcact attttcaaga ta 22
<210>169
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>169
agaccttcaa caccccagc 19
<210>170
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>170
tgtcacgcac gatttccc 18
<210>171
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>171
ccagaagtac agaaaacatg g 21
<210>172
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>172
caaggagcct atgacagtga a 21
<210>173
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>173
ctctctgctc ctcctgttc 19
<210>174
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>174
cgaccaaatc cgttgactc 19
<210>175
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>175
ctggcgtcgt gattagtgat 20
<210>176
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>176
gacgttcagt cctgtccata a 21
<210>177
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>177
cattctatca tcaacgggta caa 23
<210>178
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>178
cagcaagtgg gaaggtgtaa tc 22

Claims (7)

1. A PCR reagent for detecting apoptotic signaling pathway, comprising the following primers:
(1) the PCR reaction primers for detecting the ABL1 gene have the following primer sequences:
5'- ATCACGCCAGTCAACAGTCT -3'；
5'- TACACCCTCCCTTCGTATCT -3'；
(2) the primer sequence of the PCR reaction primer for detecting the AIFM1 gene is as follows:
5'- TCCAGCGATGGCATGTTCC -3'；
5'- CCTACTGTTGATAAGCCCACA -3'；
(3) the PCR reaction primers for detecting the AKT1 gene have the following primer sequences:
5'- GTGATCCTGGTGAAGGAGA-3'；
5'- TTAATGTGCCCGTCCTTGT-3'；
(4) the PCR reaction primers for detecting the APAF1 gene have the following primer sequences:
5'- CACACGGTTGGATCAGGAT-3'；
5'- GGAGACGGTCTTTAGCCTCT-3'；
(5) the PCR reaction primer for detecting BAD gene has the following primer sequence:
5'- CCAGAGTTTGAGCCGAGTG-3'；
5'- CCATCCCTTCGTCGTCCT-3'；
(6) the PCR reaction primers for detecting the BAG1 gene have the following primer sequences:
5'- CACACTGATCCTGCCAGAAA -3'；
5'- ACACTCGGCTAGGAATGCC -3'；
(7) the PCR reaction primers for detecting the BAG3 gene have the following primer sequences:
5'- TTCCGGTGATACACGAGCAG -3'；
5'- CTGGTGGGTCTGGTACTC -3'；
(8) the PCR reaction primers for detecting the BAK1 gene have the following primer sequences:
5'- TTTTCCGCAGCTACGTTTTT-3'；
5'- CAGAGGTAAGGTGACCATCTC-3'；
(9) the PCR reaction primer for detecting the BAX gene has the following primer sequence:
5'- GACGAACTGGACAGTAA-3'；
5'- CCTTGAGCACCAGTTT-3'；
(10) the PCR reaction primers for detecting the BCL10 gene have the following primer sequences:
5'- CAGTTGTGAACCTTTTCCAGA -3'；
5'- GGATGCCCTCAGTTTTTCAG -3'；
(11) the PCR reaction primers for detecting the BCL2 gene have the following primer sequences:
5'-AGAGCAGACGGATGGAAAAAGG-3'；
5'- GGCAAAGAAATGCAAGTGAATG-3'；
(12) the PCR reaction primers for detecting the BCL2A1 gene have the following primer sequences:
5'- ACAGGCTGGCTCAGGACTAT-3'；
5'- GCAACATTTTGTAGCACTCTG-3'；
(13) the PCR reaction primers for detecting the BCL2L1 gene have the following primer sequences:
5'- AGCTGGTGGTTGACTTTCTC-3'；
5'-CCATCTCCGATTCAGTCCCT -3'；
(14) the PCR reaction primers for detecting the BCL2L10 gene have the following primer sequences:
5'- CCAGGTTACGGCAGATTCA -3'；
5'- AAGGTCACGAGCGTCACC -3'；
(15) the PCR reaction primers for detecting the BCL2L11 gene have the following primer sequences:
5'- AAGTTCTGAGTGTGACCGAGA -3'；
5'- CTCTGTCTGTAGGGAGGTAGG -3'；
(16) the PCR reaction primers for detecting the BCL2L2 gene have the following primer sequences:
5'- CGGAGTTCACAGCTCTATAC -3'；
5'- AAAGGCCCCTACAGTTACCA -3'；
(17) the PCR reaction primers for detecting the BFAR gene have the following primer sequences:
5'- CAAGACGCCCTATACCATAGA -3'；
5'- GCCTAATGCTTTGACACGTT -3'；
(18) the PCR reaction primer for detecting BID gene has the following primer sequence:
5'- GACTGTGAGGTCAACAACG-3'；
5'- GAAGCCAAACACCAGTAGG-3'；
(19) the PCR reaction primer for detecting the BIK gene has the following primer sequence:
5'- ACCTGGACCCTATGGAGGAC -3'；
5'- CTCAGTCTGGTCGTAGATGA -3'；
(20) the PCR reaction primers for detecting the BIRC2 gene have the following primer sequences:
5'- GCACGATCTTGTCAGATTGG-3'；
5'- GCGGGGAAAGTTGAATATGTA-3'；
(21) the PCR reaction primers for detecting the BIRC3 gene have the following primer sequences:
5'-AGCTACCTCTCAGCCTACTTT -3'；
5'- CACTGTTTTCTGTACCCGGA-3'；
(22) the PCR reaction primers for detecting the BIRC5 gene have the following primer sequences:
5'- GGACCACCGCATCTCTACAT -3'；
5'-AGTCTGGCTCGTTCTCAGTG-3'；
(23) the PCR reaction primers for detecting the BIRC6 gene have the following primer sequences:
5'- GCACAGTTTCCTTGTACGGA -3'；
5'- AGCTTGGGTCTCCTGATAGAA -3'；
(24) the PCR reaction primers for detecting the BNIP2 gene have the following primer sequences:
5'- CCTAGTGATGGCTCTGTATTGT -3'；
5'- CTATTCTCTGACGGTGTGTCT -3'；
(25) the PCR reaction primers for detecting the BNIP3 gene have the following primer sequences:
5'-CAGGGCTCCTGGGTAGAACT -3'；
5'-TACTCCGTCCAGACTCATGC-3'；
(26) the PCR reaction primers for detecting the BNIP3L gene have the following primer sequences:
5'- GTCCAGTAGACCCGAAAACA -3'；
5'- GTGCTCAGTCGCTTTCCAAT -3'；
(27) the PCR reaction primer for detecting the BRAF gene has the following primer sequence:
5'- CCCAAGTCACCACAAAAACC -3'；
5'- GGACTGTAACTCCACACCTT -3'；
(28) the PCR reaction primers for detecting the CASP1 gene have the following primer sequences:
5'-TTCCGCAAGGTTCGATTTTCA -3'；
5'- GCATCTGCGCTCTACCATC-3'；
(29) the PCR reaction primers for detecting the CASP10 gene have the following primer sequences:
5'- CTTCCCAAAACTGAAATGACC -3'；
5'- CTTGATACGACTCGGCTTCC -3'；
(30) the PCR reaction primers for detecting the CASP14 gene have the following primer sequences:
5'- GCTGGAGAATCTCTTCGAGG -3'；
5'- CGTGCAAGGCATCTGTGTAT -3'；
(31) the PCR reaction primers for detecting the CASP2 gene have the following primer sequences:
5'- GCTGTTGTTGAGCGAATTGT -3'；
5'- GCAAGTTGAGGAGTTCCACA -3'；
(32) the PCR reaction primers for detecting the CASP3 gene have the following primer sequences:
5'- ATGGAAGCGAATCAATGGACT -3'；
5'-TGTACCAGACCGAGATGTCA-3'；
(33) the PCR reaction primers for detecting the CASP4 gene have the following primer sequences:
5'- CTGCGGAACTGTGCATGATG -3'；
5'- GTGTGATGAAGATAGAGCCCAT -3'；
(34) the PCR reaction primers for detecting the CASP5 gene have the following primer sequences:
5'- TCAACACCACATAACGTGTCC -3'；
5'- TCAAGGTTGCTCGTTCTATGG -3'；
(35) the PCR reaction primers for detecting the CASP6 gene have the following primer sequences:
5'- ACCAACATAACTGAGGTGGATG -3'；
5'- GGAGGAGCCATATTTTCCCA -3'；
(36) the PCR reaction primers for detecting the CASP7 gene have the following primer sequences:
5'- GGGACCGAGCTTGATGATG -3'；
5'- ACTGGGATCTTGTATCGAGGA -3'；
(37) the PCR reaction primers for detecting the CASP8 gene have the following primer sequences:
5'-TTCTGCCTACAGGTTCCACT -3'；
5'-CTCAATTCTGATCTGCTCACTT-3'；
(38) the PCR reaction primers for detecting the CASP9 gene have the following primer sequences:
5'- TGTCTACGGCACAGATGGAT -3'；
5'- GGACTCGTCTTCAGGGGAA -3'；
(39) the PCR reaction primers for detecting the CD27 gene have the following primer sequences:
5'- GCAGAGCCTTGTCGTTACAG -3'；
5'- CTCCGGTTTTCGGTAATCCT -3'；
(40) the PCR reaction primers for detecting the CD40 gene have the following primer sequences:
5'- CTGAAACGGAATGCCTTCCT -3'；
5'- CTCACTCGTACAGTGCCA-3'；
(41) the PCR reaction primers for detecting the CD40LG gene have the following primer sequences:
5'- CTCCATTTATAGCCAGCCTC -3'；
5'- TTTGGCGGAACTGTGGGT -3'；
(42) the PCR reaction primers for detecting the CD70 gene have the following primer sequences:
5'- ACCCCAGGCTATACTGGCA -3'；
5'- AGGCTGATGCTACGGGAG -3'；
(43) the PCR reaction primer for detecting the CFLAR gene has the following primer sequence:
5'-ACAGAGCTTCTTCGAGACAC-3'；
5'- CTCGGGCATACAGGCAAAT -3'；
(44) the PCR reaction primers for detecting the CIDEA gene have the following primer sequences:
5'- TTGGGAGACAACACGCATTT -3'；
5'- CTCGCTATTCCCGACCTCTT -3'；
(45) the PCR reaction primers for detecting the CIDEB gene have the following primer sequences:
5'- CAAGGACATCGCCCGATT -3'；
5'- TAGAGCCCGTAGAATGTGGC -3'；
(46) the PCR reaction primer for detecting CRDD gene has the following primer sequence:
5'- ATCAGACCGGCAGATTAACC -3'；
5'- TTGGCCTTACAGCGGTAGAT -3'；
(47) the PCR reaction primer for detecting CYCS gene has the following primer sequence:
5'- TTTGGGCGGAAGACAGGTC -3'；
5'- TATTGGCGGCTGTGTAAGAG -3'；
(48) the PCR reaction primers for detecting the DAPK1 gene have the following primer sequences:
5'- CGTGGATGATTACTACGACACC-3'；
5'- GCTTTTCTCACGGCATTTCT-3'；
(49) the primer sequence of the PCR reaction primer for detecting the DFFA gene is as follows:
5'- CCTCTTGTCAAAGCAGGAAG -3'；
5'- GTCCGAGGAGGTCTCTCT -3'；
(50) the primer sequence of the PCR reaction for detecting the DIABLO gene is as follows:
5'- GCGCAGCGTAACTTCATTC -3'；
5'- CAAAGCCAATCGTCACAGTTTT -3'；
(51) the primer sequence of the PCR primer for detecting the FADD gene is as follows:
5'- CTGGCTCGTCAGCTCAAA-3'；
5'- CTGTTGCGTTCTCCTTCTCT-3'；
(52) the primer sequence of the PCR reaction primer for detecting the FAS gene is as follows:
5'-AAGGGATTGGAATTGAGGA-3'；
5'- AGGCCTTCCAAGTTCTGAG-3'；
(53) the primer sequence of the PCR reaction primer for detecting the FASLG gene is as follows:
5'-TGCCTTGGTAGGATTGGGC-3'；
5'- CTGGTAGACTCTCGGAGTTC-3'；
(54) the PCR reaction primers for detecting the GADD45A gene have the following primer sequences:
5'- CCTGATCCAGGCGTTTTG -3'；
5'- ATCCATGTAGCGACTTTCCC -3'；
(55) the PCR reaction primers for detecting the HRK gene have the following primer sequences:
5'-GGTTCCCGTTTTCCAGAGG -3'；
5'-GCAGCTGGATTTCCAAAGG-3'；
(56) the PCR reaction primer for detecting IGF1R gene has the following primer sequence:
5'- TGCTGACCTCTGTTACCTCT -3'；
5'- GCTTATTCCCCACAATGTAGTT -3'；
(57) the primer sequence of the PCR reaction primer for detecting the IL10 gene is as follows:
5'- GACTTTAAGGGTTACCTGGGTTG-3'；
5'- TCACATGCGCCTTGATGTCTG-3'；
(58) the PCR reaction primer for detecting LTA gene has the following primer sequence:
5'- ATGACACCACCTGAACGTCTC-3'；
5'- CTCTCCAGAGCAGTGAGTTCT-3'；
(59) the PCR reaction primers for detecting the LTBR gene have the following primer sequences:
5'- CTCAGCTAAATGTAGCCGCAT-3'；
5'- ATGGTCAGGTAGTTCCAGTGC-3'；
(60) the PCR reaction primers for detecting the MCL1 gene have the following primer sequences:
5'- TGCCTTTGTGGCTAAACACT -3'；
5'- GTCCCGTTTTGTCCTTACGA -3'；
(61) the PCR reaction primer for detecting the NAIP gene has the following primer sequence:
5'- CCATTAGACGATCACACCAGA -3'；
5'- GAGTCACTTCCGCAGAGG -3'；
(62) PCR reaction primers for detecting NFKB1 gene have the following primer sequences:
5'- ACAGAGAGGATTTCGTTTCCG-3'；
5'- TTGACCTGAGGGTAAGACTTCT-3'；
(63) the primer sequence of the PCR reaction primer for detecting the NOD1 gene is as follows:
5'- CTGAAAAGCAATCGGGAACTT-3'；
5'- ACACACAATCTCCGCATCTT-3'；
(64) the primer sequence of the PCR reaction primer for detecting the NOL3 gene is as follows:
5'- ACCGCAGCTATGACCCTC -3'；
5'- TCCGGTTCAGCCTCTTTAGA -3'；
(65) the PCR reaction primer for detecting the PYCARD gene has the following primer sequence:
5'- GGATGCTCTGTACGGGAAG -3'；
5'- CAGGCTGGTGTGAAACTGAA -3'；
(66) the PCR reaction primers for detecting the RIPK2 gene have the following primer sequences:
5'- TGCCATTCACCTATGTGACA -3'；
5'- GACAGTGATGCAGCTTCATAAA -3'；
(67) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'- CTCTCTCTAATCAGCCCTCTG-3'；
5'- AGGACCTGGGAGTAGATGAG-3'；
(68) the PCR reaction primers for detecting the TNFRSF10A gene have the following primer sequences:
5'- CGGGGAGGATTGAACCAC-3'；
5'- GACGACAAACTTGAAGGTCTT-3'；
(69) the PCR reaction primers for detecting the TNFRSF10B gene have the following primer sequences:
5'- TGGAACAACGGGGACAGAAC-3'；
5'- TGCTGGGGAGCTAGGTCT-3'；
(70) the PCR reaction primers for detecting the TNFRSF11B gene have the following primer sequences:
5'- ACAAATTGCAGTGTCTTTGGTC -3'；
5'- CTGCGTTTACTTTGGTGCCA -3'；
(71) the PCR reaction primers for detecting the TNFRSF1A gene have the following primer sequences:
5'- CGCTCGTGTTTCTGGACA-3'；
5'- GTATAGACACTCGTCACTGGTG-3'；
(72) the PCR reaction primers for detecting the TNFRSF1B gene have the following primer sequences:
5'- TCATCCACGGATATTTGCAGG -3'；
5'- CTGGGGTAAGTGTACTGCC -3'；
(73) the PCR reaction primers for detecting the TNFRSF21 gene have the following primer sequences:
5'- TGACTGACCGAGAATGCACT -3'；
5'- TCATCACACTAGAAGGCACATC -3'；
(74) the PCR reaction primers for detecting the TNFRSF25 gene have the following primer sequences:
5'- TGGCAGCGGGCACATTCAGAATT-3'；
5'- CTTGCACGGAGCCCTGCGGCAA-3'；
(75) the PCR reaction primers for detecting the TNFRSF9 gene have the following primer sequences:
5'- TGGATGGAAAGTCTGTGCTTG -3'；
5'- GGAGATGATCTGCGGAGAGT -3'；
(76) the PCR reaction primers for detecting the TNFSF10 gene have the following primer sequences:
5'- CCACCCTGACCTACACATAC-3'；
5'- TTACCCACCAACTGGACGG-3'；
(77) the PCR reaction primers for detecting the TNFSF8 gene have the following primer sequences:
5'- GAGGACGGACTCCATTCCC -3'；
5'- GTAGGCCCATGACTTCTTGAA -3'；
(78) the primer sequence of the PCR reaction primer for detecting the TP53 gene is as follows:
5'- AGCACATGACGGAGGTTGT-3'；
5'- CATCCAAATACTCCACACGC-3'；
(79) the primer sequence of the PCR reaction primer for detecting the TP53BP2 gene is as follows:
5'- AAGTGCGTCCGTTCTCAATG -3'；
5'- GTTCTTCCTCAGAGTACCAAAG -3'；
(80) the primer sequence of the PCR reaction primer for detecting the TP73 gene is as follows:
5'- ACGAGGACACGTACTACCTT -3'；
5'- TGCCGATAGGAGTCCACCA -3'；
(81) the PCR reaction primer for detecting the TRADD gene has the following primer sequence:
5'- ATTCTGCCTCAGGTACTT-3'；
5'- CAGGACACCAAAGATCAA-3'；
(82) the primer sequence of the PCR reaction primer for detecting the TRAF2 gene is as follows:
5'- CCCTGGAGTTGCTACAGC-3'；
5'- GGCGGAGCACAGGTACTT -3'；
(83) the primer sequence of the PCR reaction primer for detecting the TRAF3 gene is as follows:
5'- AGACTAACCCGCCGCTAAAG-3'；
5'- ATGCTCTCTTGACACGCTGT-3'；
(84) the primer sequence of the PCR reaction primer for detecting the XIAP gene is as follows:
5'- CCGTGCGGTGCTTTAGTT-3'；
5'-GCGTGGCACTATTTTCAAGATA-3'；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'- AGACCTTCAACACCCCAGC-3'；
5'- TGTCACGCACGATTTCCC-3'；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'-CCAGAAGTACAGAAAACATGG-3'；
5'-CAAGGAGCCTATGACAGTGAA-3'；
(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'-CTCTCTGCTCCTCCTGTTC-3'；
5'-CGACCAAATCCGTTGACTC-3'；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'-CTGGCGTCGTGATTAGTGAT-3'；
5'-GACGTTCAGTCCTGTCCATAA-3'；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'-CATTCTATCATCAACGGGTACAA-3'；
5'-CAGCAAGTGGGAAGGTGTAATC-3'。
2. use of the PCR reagent of claim 1 for the preparation of a kit for detecting apoptotic signaling pathway.
3. Use of a PCR reagent according to claim 2 in the preparation of a kit for detecting apoptotic signaling pathways, wherein: the cells comprise cancer cells and normal cells, and the cancer cells are oral squamous carcinoma cells, breast cancer cells, lung cancer cells and liver cancer cells.
4. Use of a PCR reagent according to claim 2 in the preparation of a kit for detecting apoptotic signaling pathways, wherein: the kit uses a real-time fluorescent quantitative PCR detection method for detection.
5. The use of the PCR reagent according to claim 4 for the preparation of a kit for detecting apoptotic signaling pathway, wherein: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1 mul-0.2 mul of upstream primer, 0.1 mul-0.2 mul of downstream primer, 25ng-100ng of cDNA template, and sterile distilled water to make up to 20 mul.
6. Use of a PCR reagent according to claim 5 in the preparation of a kit for detecting apoptotic signaling pathways, wherein: the Taq polymerase is 2X SYBR Premix Ex Taq polymerase of TaKaRa company.
7. The use of the PCR reagent according to claim 4 for the preparation of a kit for detecting apoptotic signaling pathway, wherein: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 deg.C for 5 seconds, 59 deg.C for 20 seconds, and 40 cycles; the dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
CN202010666818.2A 2020-07-13 2020-07-13 PCR reagent for detecting apoptosis signal path and application thereof Pending CN111808958A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005107782A2 (en) * 2004-05-11 2005-11-17 The University Of Nottingham Use of a modulator of gene expression in the treatment of cancer
CN101084914A (en) * 2007-06-22 2007-12-12 南京中医药大学 Application of ophiopognin saponin D in preparing vascular endothelial cells apoptosis regulating gene medicine
CN102251050A (en) * 2011-08-10 2011-11-23 青岛大学医学院附属医院 Reagent for detecting cell apoptosis signal path and PCR method using same
CN104450886A (en) * 2014-11-03 2015-03-25 青岛大学附属医院 PCR reagent for detecting NF-kappa B signaling pathways in cells and application of PCR reagent

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005107782A2 (en) * 2004-05-11 2005-11-17 The University Of Nottingham Use of a modulator of gene expression in the treatment of cancer
CN101084914A (en) * 2007-06-22 2007-12-12 南京中医药大学 Application of ophiopognin saponin D in preparing vascular endothelial cells apoptosis regulating gene medicine
CN102251050A (en) * 2011-08-10 2011-11-23 青岛大学医学院附属医院 Reagent for detecting cell apoptosis signal path and PCR method using same
CN104450886A (en) * 2014-11-03 2015-03-25 青岛大学附属医院 PCR reagent for detecting NF-kappa B signaling pathways in cells and application of PCR reagent

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Title
CAT.NO.330231 PAHS-012ZA: "RT2 Profiler PCR Array (96-Well Format and 384-Well [4×96] Format) Human Apoptosis", 《QIAGEN》 *
L. LO MUZIO等: "Genetic Analysis of Oral Squamous Cell Carcinoma by cDNA Microarrays Focused Apoptotic Pathway", 《INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY》 *

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