SYBR实时荧光定量PCR检测人乳腺癌细胞AnnexinⅡmRNA方法的建立及应用-论文.pdf

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1、重庆医学2014年6月第43卷第18期2323·技术与方法·SYBR实时荧光定量PCR检测人乳腺癌细胞AnnexinⅡInRNA方法的建立及应用郭变琴，黄学梅，吴立翔△(重庆市肿瘤研究所检验科400032)摘要：目的建立检测人AnnexinIItuRNA水平的SYBRGreen实时荧光定量PCR方法，并用其测定人乳腺癌细胞MDA—M231和MCF-7中AnnexinⅡmRNA水平。方法根据人AnnexinIImRNA的保守序列设计特异性引物，提取人乳腺癌细胞总RNA，并逆转录为cDNA，胶回收纯化PCR产物

2、，并与pGM-T载体连接构建质粒标准品，进一步通过sYBRGreen实时荧光定量PCR检测人乳腺癌细胞中AnnexinlImRNA水平。结果该方法标准曲线的r2为0．997，融解曲线分析显示单个峰，pGTAnnexinlI质粒标准品高、低拷贝数的批内、批问变异系数分别为6．2、7．8(高拷贝)和9．1、12．3(低拷贝)。进一步研究表明人乳腺癌细胞MDA—M231中AnnexinⅡmRNA水平显著高于MC7细胞(P<0．01)。结论建立的检测人AnnexinIImRNA水平的SYBRGreen实时荧光定量P

3、CR方法特异性、重复性好，可用于人乳腺癌细胞中AnnexinⅡmRNA的定量检测。关键词：乳腺肿瘤；AnnexinⅡ；SYBRGreen实时荧光定量PCRdoi：10．3969／j．issn．1671—8348．2014．18．021文献标识码：A文章编号：1671—8348(2014)18-2323—03EstablishmentandapplicationofSYBRrealtimefluorescencequantitativePCRfordetectingAnnexinⅡmRNAofhumanbre

4、astcancercellsGuoBianqin，HuangXuemei，WuLixiang(DepartmentofClinicalLaboratory，ChongqingCancerInstitute，Chongqing400032，China)Abstract：ObjectiveToestablishaSYBRGreenbasedreal—timefluorescencequantitativePCRmethodfordetectinghumanAnnexinIImRNAexpression．andt

5、odetectthelevelofAnnexinIImRNAinhumanbreastcancercellsMCF_7andMDA—M231．MethodsThespecificprimersweredesignedaccordingtotheconservedsequenceofhumanAnnexinⅡgene．TotalRNAswereextractedfromhumanbreastcancercells(MCF_7，MDA_-M231)，thenRNAsweretranscribedreversel

6、yintocDNAs．Theplasmidstandardswereconstructed．TherelativeexpressionlevelsofhumanAnnexinIImRNAinhumanbreastcancerceilsweredetectedbythismethod．ResultsThesquare()ofcorrelationcoefficientofthestandardcurveinthismethodwas0．997，themeltingcurveanalysisshowedthes

7、inglepeak．Thetheintra—batchandinter-batchvariablecoefficientsinthepGM-TAnnexinⅡplasmidstandardsubstancewere6．2，7．8and9．1，12．3respectively．ThefurtherstudyindicatedthatAnnexinIImRNAinMDA—MB-231washigherthanthatinMCF-7(P

8、nrealtimefluorescencequan—titativePCRmethodfordetectinghumanAnnexinIIisofgoodspecificityandrepeatabilityandcanbeusedforquantitativelydetec—tingAnnexinlImRNAinbreastcancercells．Keywords：breastneoplasms；Annexin