第30卷第5期西安交通大学学报(医学版)Vol.30No.52009年10月JournalofXipanJiaotongUniversity(MedicalSciences)Oct.2009利用TRIzoló试剂和液氮提取大鼠胰腺高质量总RNA李冬民1,2,任吴超1,2,王璇1,2,王飞苗1,2,高玉1,2,韩燕1,2,宁启兰1,2,宋天保1,3,吕社民1,2(西安交通大学医学院:1.环境与疾病相关基因教育部重点实验室;2.遗传学与分子生物学系;3.人体解剖学与组织胚胎学系,陕西西安710061)摘要:目的建立胰腺高质量总RNA快速、经济、稳定的提取方法。方法应用TRIzoló试剂和液氮提取大鼠胰腺总RNA,通过总RNA含量和A260/280比值的测定及10g/L非变性琼脂糖凝胶电泳评估总RNA的质量,并用RT2PCR检测大鼠胰岛素1、胰高血糖素、胰α2淀粉酶和β2actin的表达。结果利用TRIzoló和液氮提取的大鼠胰腺总RNA量可达到3～6μg/mg胰腺组织,A260/280比值在1.75～1.89之间。在10g/L非变性琼脂糖凝胶电泳时,均可见28S及18S条带,并且在28S、18S条带间可见明显的云雾状阴影。大鼠胰岛素1、胰高血糖素、胰α2淀粉酶、β2actinRT2PCR产物电泳结果显示均有清晰的条带。结论应用TRIzoló试剂和液氮成功地提取了大鼠胰腺高质量的总RNA,后者可用于RT2PCR研究。关键词:大鼠胰腺;总RNA提取;TRIzoló试剂;液氮中图分类号:Q813文献标志码:A文章编号:167128259(2009)0520639204,RENWu2chao1,2,WANGXuan1,2,WANGFei2miao1,2HANYan1,2,NINGQi2lan1,2,SONGTian2bao1,3(1.KeyLaboratoryofEnvironmentandGenesRelatedtoDiseases,MinistryofEducationofChina/AmethodwithTRIzolóreagentandliquidnitrogentoextracthigh2qualityRNAfromratpancreasLIDong2min1,2,GAOYu1,2,,LBShe2min1,2MedicalSchoolofXipanJiaotongUniversity,Xipan710061;2.DepartmentofGeneticsandMolecularBiology,MedicalSchoolofXipanJiaotongUniversity,Xipan710061;3.DepartmentofHumanAnatomy,HistologyandEmbryology,MedicalSchoolofXipanJiaotongUniversity,Xipan710061,China)ABSTRACT:ObjectiveToestablishaquick,economicalandreproduciblemethodforhigh2qualityRNAextractionfrompancreas.MethodsWeutilizedTRIzolóReagentandliquidnitrogentoisolatetotalRNAfromtheratpancreas.TheRNAqualitywasdeterminedbydetectionofitscontentandopticdensity(A)at260/280nm,andelectrophoresisin1%non2denaturedagarosegel.Thenreversetranscription2polymerasechainreaction(RT2PCR)wasperformedtodetectexpressionofthepancreas2specificgenes.ResultsThecontentofthetotalRNAextractedfromtheratpancreasreached3-6μg/mgpancreatictissues,andA260/280ratiowas1.75-1.89.ElectrophoresisofthetotalRNAshowed28Sand18SrRNAbandswithclearsmearbetweenthem.TheRT2PCRproductsofpancreas2specificgenesincludinginsulin1,glucagon,α2amylaseandhousekeepinggeneβ2actinallexhibitedclearbandson1%agarosegel,whichwerelocatedintheexpectedpositions,respectively.ConclusionTheseresultssuggestthatwehavesuccessfullyisolatedthehigh2qualityandintactRNAfromtheratpancreaswithTRIzolóReagentandliquidnitrogen.TheextractedtotalRNAcanbeusedinRT2PCRforpancreaticgeneexpres2sion.KEYWORDS:ratpancreas;totalRNAextraction;TRIzolóreagent;liquidnitrogen收稿日期:2008212223修回日期:2009203218基金项目:国家自然科学基金资助项目(No.30400249;30571725;30630058);陕西省国际科技合作重点项目(No.20072KW206);陕西省自然科学基金资助项目(No.2004C256);陕西省卫生厅基金资助课题(No.D204028)SupportedbytheNationalNaturalScienceFoundationofChina(No.30400249;30571725;30630058),theInternationalCooperationalFoundationofShaanxiProvince(No.20072KW206),theNaturalScienceFoundationofShaanxiProvince(No.2004C256),andtheFoundationofHealthDepartmentofShaanxiProvince(No.D204028).通讯作者:宋天保,教授,博士生导师.E2mail:songtbao@mail.xjtu.edu.cn;吕社民,教授,博士生导师.E2mail:lushemin@mail.xjtu.edu.cn作者简介:李...