低密度cDNA 芯片杂交条件的优化及验证分析

(110001#110001)cDNAcDNA(1414)1891078bp0.5µg/µLSGC7901(16121824h)(37506068)2RT-PCRSGC790118h24h(P0.001)60506837(P0.001)2(t=-0.941P=0.348)0.588RT-PCR-0.778(P0.0001)RT-PCR100%RT-PCR201680%(16/20)6018hcDNART-PCRcDNAOptimizationofLow-densitycDNAMicroarrayHybridizingConditionandVerificationAnalysisZhaoYujie,HuangBaojun#,XuHuimian#,HeQun(TheCenterofBiochip,ChinaMedicalUniversity,Shenyang110001#OncologyDepartment,TheFirstAffiliatedHospital,ChinaMedicalUniversity,Shenyang110001)AbstractInordertooptimizethebesthybridizingconditionoflow-densitycDNAmicroarrayestablishedbytheauthorsandtofurtherdiscussthereproducibilityandreliability,alow-densitycDNAmicroarray(14×14)wasfabricated,onwhichthetargetgenes’lengthwasbetween189and1078basepairs,andtheconcentrationwasabove0.5µg/µL.SGC7901gastriccancercellswereusedfordetection.Differenthybridizingtime(1h6h12h18h24h)andtemperature(37506068)weredesignedtocomparethehybridizingdynamics.Alltargetgenes’mRNAexpressioninthesamecellsweredetectedbytheidenticalcDNAmicroarraytwiceandbyRT-PCRforfurtherprovingtoknowthereproducibilityandreliability.Thelongerofhybridizingtime,thestrongerofsignalintensity,atimeof18hwasthebestandsignalintensitydecreasedafter24h(P0.001).Withrespecttothehybridizingtemperature,60wasthestrongest,andbecamelessat506837onebyone(P0.001).Therewasnosignificantdifferenceintworepeateddetection(t=0.941P=0.348),andthecorrelationcoefficientwas0.588.IncomparisonwithRT-PCR,thecorrelationcoefficientwas–0.778(P0.0001),specificitywas100%andsensitivity80%.In45(2001101001)2004-06-292004-11-01[1~6]cDNA()[7~9]cDNA(200bp)(1000bp)50%DMSO0.5µg/µL[10]cDNA11.1MicroGrind()GeneTAC()PCR(WhatmanBiometra)ChemiImager5500UV300N-Cy5-dUTP(Pharmacia)SuperscriptreversetranscriptaseAMV(Promega)TRIzol(Invitrogen)TaqDNA()()(CEL)1.21.2.1cDNAbetaactinGAPDHHeparanaseMMP-2MMP-7VEGF-CS100A4hRad17hTERTTGF-βCD44sIntegrin-βCDH1KAI1TIMP-1TIMP-2PTENnm23H1DNAPrimerPremier5.0RT-PCRPCR1891078bp1betaactinGAPDHDNA50%DMSORT-PCR(RNA)PCR(DNA)50%DMSO0.5µg/µL1414[10,11]1.2.2SGC7901TRIzolRNARNA1%RNA100µgcDNACy5EDTANaOH[10,11]1RT-PCRPCRTab.1TheRT-PCRandPCRprimers,lengthofproductsforallthegenes/bp/bpactinB15`-CACCAACTGGGACGACAT-3`MMP-75`-AGATGTGGAGTGCCAGATGT-3`5`-ACAGCCTGGATAGCAACG-3`1895`-TAGACTGCTACCATCCGTCC-3`358actinB25`-GATGACCCAGATCATGTTTG-3`MMP-25`-TCCCCGATAACCTGGATGC-3`5`-TGGAGTTGAAGGTAGTTTCG-3`4915`-CTCACTCTTACGACTAATCGACATCT-3`276actinB35`-TCCTATGTGGGCGACGAG-3`TIMP15`-GCCCCTGGCTTCTGGCATCCT-3`5`-TAGAAGCATTTGCGGTGG-3`9745`-GAGGCAGGCAGGCAAGGTGAC-3`559GAPDH15`-CGACCACTTTGTCAAGCTCA-3`TIMP25'-CCCCATGATCCCGTGCTA-3`5`-AGGGGTCTACATGGCAACTG-3`2275`-CGACCTGGTCAGCTTTGGG-3`252GAPDH25`-GGGAAACTGTGGCGTGAT-3`TGFβ5`-AAGTGGATCCACGAGCCCAA-3`5`-TGGCAACTGTGAGGAGGG-3`5525`-GTCGCACTTGCAGGAGCGCA-3`243GAPDH35`-GGTCACCAGGGCTGCTTT-3`CD44S5`-GACACATATTGCTTCAATGCTTCAGC-3`5`-TGGCAACTGTGAGGAGGG-3`10785`-GATGCCAAGATGATCAGCCATTCTGGAA-3`482HBV5`-GTGCCATTTGTTCAGTG-3`nm23H15`-CGCCTTGTGGGTCTGAAA-3`5`-ATACTTTCCAATCAATAGG-3`305(PCR)5`-TGTGGTCTGCCCTCCTGT-3`377hTERT5`-GGCGACATGGAGAACAAGC-3`KAI15`-AGCCTGTATCAAAGTCACCAA-3`5`-AGGTGAGACTGGCTCTGATGG-3`3405`-GCAGAAGCCCTTCCTCACA-3`571VEGF-C5`-GAGGCTGGCAACATAACAGAG-3`CDH15`-CCCCATACCAGAACCTCG-3`5`-CCTTGAGAGAGAGGCACTGT-3`3215`-ACCGCTTCCTTCATAGTCAA-3`742hRad175`-AAAATCAAGAGGTCCAAG-3`pten5'-CATACCAGGACCAGAGGAAACC-3`5`-CCTGAGTAAAGAGCGTGT-3`3355`-ACCTAGTCTCAGTCACCACAGTC-3`325S100A45`-CCTGGATGTGATGGTGTC-3`Integrinβ5`-GTGGAGAATGTATACAAGCAGG-3`5`-TCTTCCTGGGCTGCTTAT-3`2785`-CTAATGTAAGGCATCACAGTC-3`486heparanase5`-AGAAAGACGGCTAAGATG-3`5`-ATAGGGTAACCGCAAGTA-3`5881.2.3betaactin(189491974bp)GAPDH(2275521078bp)50%DMSO0.5µg/µL320SGC790121.2.3.16016121824h1.2.3.218h375060681.2.4cDNA2(3%8%)ChemiImager5500(A)025521.2.6SPSS11.5tRT-PCR22.1RNA12DL2000100bpmarker50ngA2600.5µg/µLSGC7901RNA1%A260/A28018S28S1/(22.5)A260/A2801.8RNA1Fig.1ThepurifiedtargetgenesbyployacrylamidegelelectrophoresisandsilverdyeingActin1,Actin2,Actin3,GAPDH1,2,3,Marker,Integrin-β,VEGF-C,TGF-b,Heparanase,hTERT2Fig.2ThepurifiedtargetgenesbyployacrylamidegelelectrophoresisandsilverdyeingHBV,TIMP-1,MMP-7,MMP-2,TIMP-2,Marker,S100A4,hRad17,CD44s,CDH1,PTEN,KAI1,nm23H12.22.2.11h6121824h18h24h12h(P=0.422)(P0.0001)36024h2.2.23768506060(P0.0001)418h’signalintensitymeanvaluesatdifferenthybridizationtime4Fig.4Comparisonofallgenes’signalintensitymeanvaluesatdifferenthybridizationtemperature2.3cDNASGC79016018h220.588(P=0.0000)t(t=-0.941P=0.348)55SGC7901(Cy5)Fig.5HybridizationofSGC7901gastriccancercells(Cy5labeled)2.4cDNAmRNART-PCRRT-PCR0.778(P0.0001)RT-PCRRT-PCR100%RT-PCR201680%(16/20)3