冰冻组织特定区域的RNA快速提取及其RT-PCR分析

RNART-PCR江岩　刘宁　刘佳　李宏RNA,PCR(reversetranscription-PCR,RT-PCR)RNART-PCR,5m20m,,20m,RNART-PCR1RNA,6RT-PCRRNA,,RNART-PCR;;RNA;QuickRNAextractionfromspecifiedmorphologicalregioninfrozensectionsanditsRT-PCRapplication*JANGYan,LIUNing,LIUJia,LIHong.(DivisionofMolecularBiology,ShenyangMedicalCollege,Shenyang,Liaoning,110031P.R.China.E-mail:[e-mail])AbstractObjectiveToestablishamethodforquickRNAextractionfromspecifiedhistologicalregioninfrozensectionssoastoavoidcrossRNAcontaminationamongdifferenttypesofcells/tissuesandtoachievespecificRT-PCRamplification.MethodsPrepareserialfrozensections5minthicknessforhis-tologicalandimmunocytochemicalexamination.Basedontheresults,makeoneortwopiecesof20msec-tionandselectaspecifiedregionforquickRNAextractionandRT-PCRanalysis.ResultsRNAextractioncanbefinishedwithinanhourandthedataofRT-PCRobtainedwithin6hours.Besidesthehighperfor-mance,thismethodcanefficientlyexcludepseudopositiveorpseudonegativeresultscausedbyinter-cellu-larRNAcontamination.ConclusionThismethodisanimportantcomplementtothecurrenttechniquesforRNAextractionandRT-PCR.Keywordsfrozensection;specifiedhistology;quickRNAextraction;reversetranscription-polymerasechainreaction*ProjectNo.963010,supportedbySpecialGrantforOutstandingYoungScientistsfromLiaoningCommitteeofSci-ence&TechnologyRNA(reversetran-scription-polymerasechainreaction,RT-PCR)[1],RNA(RNase),RNA,RNA,RNA,RNA60%80%,RNA,():(9663010):110031:(E-mail:[e-mail]),RNA,,,,,,RNART-PCR11.11h,,-751.2RNAA(solutionA)[2]:1.25PCR(Takara,);5U4720002171ChinJMedGenet,February2000,Vol.17,No.1RNasin1URNasefreeDNase(Promega,USA);2mmol/LDTT,Superscriptreversetranscriptase5Firststrandbuffer(GibcoBRL,USA);TaqDNAPolymerase,10PCRdNTPs(Takara,)PCR(receptorofhyaluronicacidmediatedmotili-ty,RHAMM);PCROligo,:5:5-AGCTGGCCGTCAACAT-GTCC;3:3-CCCTTGAGACTCTTCGAGACT-CC1.3LeicaCMI800100%0.1%,RNase;-24,5mH/E,,(),Ze-bra,-20,RHAMM(V.Assman)ABC[3]1.4RNAH/E,,,20m,;20lA500l,;371h,DNA;9515min,DNase;13600r/min5min,16lRT-PCR1.5RT-PCRJiang[2]cDNAPCR,RT-PCR:3.5l6.5lRT(1firststrandbuffer,2mmol/LdNTP,10mmol/LDTT,100UsuperscriptTMreversetran-scriptase,50pmol/LRHAMM-actin)RT371hRT955min10RT2.5l165lPCR,6.5lPCR(1PCR,1mmol/LdNTP,50pmol/LRHAMM-actin,2.5UTaqDNA)PCR:9430s,6030s,7235s,3040;,755minPCR0.5g/ml0.014174HaeDNA(Promega,USA)22.131,6968H/E,(1a);(1b);,,,(1cd)2.2RHAMM:RHAMM(1a);(1b);,RHAMM(1cd)2.3RNART-PCR,RNARHAMMRT-PCRICC(2)RNART-PCR,(2),Jiang,PCR(2)(3B)RTPCR,RNARTPCR,(3C)3PCR10,,(DNARNA)PCR,PCR,,,,,/DNARNADNA[4,5]RNA,1995Jiang[2]RNA,RT-PCRRT-PCR,RNA,RNA,Jiang4820002171ChinJMedGenet,February2000,Vol.17,No.11RHAMMa:;b:;c:;d:Fig1HistologicalandRHAMMimmunocytochemicalstainingonthefrozensectionsa:inflammatorygastricmucosa;b:Gradedysplasiawithintestinalmetaplasia;c:thegastriccancertissuewithoutdifferentiation;d:moderatelydifferentiatedsub-populations21RNAJiangRHAMMRT-PCR33RNARHAMMRT-PCRA:,RTPCR;B:Jiang;C:RT-PCRFig2BytheuseofJiang'sprotocol,RT-PCRforRHAMMexpressionwasperformedontheRNAsamplesquick-extractedfromthespecifiedhisto-logicalregionsshowninFig1Fig3GelelectrophoresisofRHAMMRT-PCRproductsamplifiedbydifferentexperimentalapproachesfromthequick-extractedRNAsamplesof3stomachcancersA:PCRwithoutRT;B:Jiang'sprotocol;C:RT-PCRwiththeroutinepreparationsRNA,RHAMM,,Jiang,:(1),20m2mm2mm(2),,1/4RNA,1/10RT,304050PCR,,RNART-PCR(3)RNA,RTPCRJiang,,,RTPCRJiang,,(),4920002171ChinJMedGenet,February2000,Vol.17,No.1RNA,PCRRNA,,1GaneshVR,JoseGM,LeonardGG.Utilizationofpolymerasechainreactiontechnologyinthedetectionofsolidtumors.Cancer,1998,82(8)1419-1441.2JiangYH,DavidsonLA,LuptonJR,etal.ArapidRT-PCRmethodfordetectionofintactRNAinformalin-fixedparaffin-embeddedtis-sues.NucleicAcidRes,1995,23(15)3071-3072.3LiH,HamouMF,deTriboletN,etal.VariantCD44adhesionmoleculesareexpressedinhumanbrainmetastasesbutnotinglioblastomas.CancerRes,1993,53(22)5345-5349.4KumarR,BarbacidM.Oncogenedetectionatthesinglecelllevel.Oncogene,1988,3(6)647-651.5LiH,LeeGH,LiuJ,etal.AbsenceofH-raspointmutationatcodon12inMNU-inducedhepatocellularcarcinomasintherat.JCancerResClinOncol,1989,116(4)331-335.(:1999-03-06)()血管脂肪瘤一家系八例于慧敏　许贵存　赵晓东　朱秀兰(8),3519,,,,,,:,,,,,,,550mm,,,40::,25,,7,182616,39,2,24,,,1,,,,,,,,,:010050,1,,,,7,3,,,1/2,,31(:1999-03-03)()5020002171ChinJMedGenet,February2000,Vol.17,No.1