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1、ChinJNeurosci,1999;Vol15(2):151～155151技术方法1513银染mRNA差异显示方法的条件优化331梁德勇 王晓民 崔振中 徐国恒 朱丽霞 韩济生(1北京医科大学神经科学研究所 北京 100083;中国中医研究院针灸研究所 北京 100700)摘要 mRNA差异显示技术是分离差异表达基因的强有力工具。本工作旨在优化无同位素的银染差异显示方法。以总RNA为模板,用锚定引物与随机引物的组合进行RT2PCR反应,PCR扩增产物在变性的聚丙烯酰胺凝胶上电泳。用银染方法显示电泳cDAN条带,其结果是:(1)RNA加入量
2、为1～3Lg,RT2PCR反应能扩增出较多的cDNA条带,而低于015Lg时扩增条带数目较少;(2)在36～42℃的范围内,随着温度的增加,扩增的条带减少;而42℃时扩增的条带数目锐减,特别是当RNA加入量小于1Lg时几乎无条带显示;36℃扩增的条带过多而趋于smear;(3)优化了银染液程序,染色液和显色液中37%甲醛的量分别为0103%～0105%和01075%～011%,显色温度4～12℃时,显示出清晰的条带;(4)用此方法获得数条电针镇痛有效与无效大鼠表达差异的cDNA条带。总之,通过改变参数条件,优化了快速简便的银染mRNA差异显
3、示方法。关键词 mRNA差异显示 逆转录2聚合酶链反应 银染 基因表达OPTIMIZATIONOFmRNADIFFERENTIALDISPLAYWITHSILVERSTAINING1LIANGDe2Yong,WANGXiao2Min,CUIZhen2Zhong,XUGuo2Heng,ZHULi2Xia,HANJi2Sheng(1NeuroscienceResearchInstitute,BeijingMedicalUniversity,Beijing100083;TheInstituteofAcupunctureandMoxibustion
4、,ChinaAcademyofTraditionalChineseMedicine,Beijing100700)ABSTRACTmRNAdifferentialdisplayisapowerfulinstrumenttoidentifydifferentiallyexpressedgenes.Thisworkisdesignedtooptimizeanon2radioactivedifferentialdisplaywithsilverstaining.TotalRNAastemplatewasreversetranscribedintoc
5、orrespondingcDNAsby3′anchorprimerandthecDNAswereamplifiedbypolymerasechainreaction(PCR)withasetofonesame3′anchorprimerandone5′arbitrarilyprimer.ThePCRproductswerethenresolvedondenaturingpolyacylamimidegel.cDNAbandswerevisualizedbysilverstaining.Theresultsrevealedasfollows:
6、(1)TheamountofRNAusedwas1～3Lg,whichproducedmanycDNAbandsbyRT2PCR.FewofbandswereobservedwhentheamountofRNAwasreducedtolessthan0.5Lg.(2)cDNAbandsweredecreasedwithincreasingtemperatureinarangeof36～42℃.(3)Silverstainingprocedurewasmodified.Theconcentrationof37%formaldehydeinst
7、ainingsolutionanddevelopingsolutionwasoptimized,andthedevelopingtemperaturewasappropriateat4～12℃.(4)SeveralcDNAfragmentswereidentifiedfrombrainofresponderandnon2responderratforelectroacupu2ncture2inducedanalgesia.Inconclusion,thesilverstainingmRNAdifferentialdisplayoptimiz
8、edinparametersisarapidandeasyprocedure.3国家自然科学基金(No.39600192)资助课题 33联系人Received1998211
