实时荧光定量pcr检测人il 9 mrna方法的建立及应用

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1、实时荧光定量PCR检测人IL9mRNA方法的建立及应用来源：创新医学网 　　作者：杨先知1,史烨1,柳迎昭2,陈建　　作者单位：1.江苏大学基础医学与医学技术学院免疫学和免疫检验学系,江苏镇江212013;2.江苏大学附属人民医院内科，3.检验科，江苏镇江212002　　【摘要】目的：建立检测人白介素9(interleukin9，IL9)mRNA的SYBRGreenⅠ实时荧光定量PCR(realtimefluorescencequantitativePCR,qRTPCR)方法。方法：分离人外周血单个核细胞(

2、peripheralbloodmononuclearcells,PBMC)，经PMA和离子霉素刺激活化后提取总RNA并逆转录成cDNA。扩增产物与pMD18T载体连接构建质粒标准品。采用qRTPCR方法检测IL9mRNA的表达水平。评价该方法的线性及特异性，应用该方法检测Graves病患者PBMC中IL9mRNA表达水平。结果：建立了人IL9的SYBRGreenⅠ实时荧光定量PCR检测方法。该法的标准曲线相关系数r2为0.995～0.998，熔解曲线分析产物为单峰。Graves病患者PBMC中IL9mRN

3、A的相对表达水平明显高于健康对照组(P<0.01)。结论：建立的检测人IL9mRNA的SYBRGreenⅠ实时荧光定量PCR方法灵敏、特异性好，为进一步临床应用提供了实验基础。　　【关键词】白细胞介素9;实时荧光定量PCR;SYBRGreenⅠ　　Establishmentandapplicationoftherealtimefluorescencequantitative　　PCRassayfordetectionhumanIL9mRNAexpression　　YANGXianzhi1,SHIYe1,LI

4、UYingzhao2,CHENJianguo2,TIANJie1,LIUYan1,LIUQingling1,　　SUZhaoliang1,TONGJia1,MABin1,SHAOQixiang1,XUHuaxi1,WANGShengjun1　　(1.DepartmentofImmunology,SchoolofMedicalScienceandLaboratoryMedicine,JiangsuUniversity,ZhenjiangJiangsu212013;2.DepartmentofMedicin

5、e;3.DepartmentofClinicalLaboratory,theAffiliatedPeople′sHospitalofJiangsuUniversity,ZhenjiangJiangsu212002,China)　　[Abstract]Objective:ToestablishaSYBRGreenⅠbasedrealtimefluorescencequantitativePCR(qRTPCR)methodfordetectionhumaninterleukin9(IL9)mRNAexpressi

6、on.Method:ThespecificprimersweredesignedaccordingtotheconservedsequenceofhumanIL9gene.TotalRNAswereextractedfromhumanperipheralbloodmononuclearcells(PBMC)whichwerestimulatedbyPMAandionomycin,thentheRNAsweretranscribedreverselyintocDNAs.Theplasmidstandardswerec

7、onstructed.Thesensitivityandspecificityofthemethodwereevaluated.TherelativeexpressionlevelsofhumanIL9mRNAinPBMCsfrompatientswithGraves′disease(GD)andhealthycontrolsweredetectedbythismethod.Results:Thecoefficientofcorrelationofthestandardcurveofthismethodwas0.9

8、95-0.998,meltingcurveanalysisshowedsinglepeak.TherelativeexpressionlevelsofhumanIL9mRNAinGDgroupwerehigherthanthatinhealthycontrolgroup(P<0.01).Conclusion:locatedintheTomb,