ResearchpaperCharacterizationofspecificcDNAbackgroundsynthesisintroducedbyreversetranscriptioninRT-PCRassaysM.F.Adrovera,M.J.Muñozb,M.V.Baeza,J.Thomasc,A.R.Kornblihttb,1,A.L.Epsteinc,1,D.A.Jerusalinskya,*,1aInstitutodeBiologíaCelularyNeurociencias“Prof.EduardoDeRobertis”IBCN-CONICET-FacultaddeMedicina,UniversidaddeBuenosAires,Paraguay2155,2dopiso(1121)CiudaddeBuenosAires,ArgentinabLaboratoriodeFisiologíayBiologíaMolecular,DepartamentodeFisiología,BiologíaMolecularyCelular,IFIBYNE,CONICET-FacultaddeCienciasExactasyNaturales,UniversidaddeBuenosAires,ArgentinacCentredeGénétiqueMoléculaireetCellulaire,UniversitéClaudeBernardLyon1,CNRS-UMR5534,Villeurbanne,FrancearticleinfoArticlehistory:Received12April2010Accepted30July2010Availableonline13August2010Keywords:ReversetranscriptionAntisenseRNABackgroundsynthesisRT-PCRHSV-1vectorsabstractToblockexpressionofNMDAreceptorNR1subunit,weinjectedintorathippocampusaHerpesSimplexVirustype1derivedvectorbearingasequenceforNR1antisense.RT-PCRassayswithRNAfromhippocampusofanimalsinjectedeitherwithNR1antisensevector,controlvectororvehicle,showedanamplificationsignalcompatiblewithNR1antisensewhichcouldbeattributedeithertoanendogenousNR1antisenseortoanartifact.RT-PCRwasperformedeitherwithdifferentprimersorwithoutprimersintheRT,usingRNAfromdifferenttissues.RNAseprotectionassaywascarriedouttocharacterizetheamplifiedsignalnature.OurresultsshowthatthetemplatefortheunexpectedamplifiedfragmentwasNR1mRNAcurrentlyexpressedinnervoustissue.WeconsideredthisbasalamplificationofamRNAinaRT-PCRassayas“backgroundamplification”.Afterbackgroundsubtraction,asignificantsignalonlyremainedwhensamplesfromNR1antisensevectorinjectedanimalswereused,demonstratingthatthiswastheonlysourceforNR1antisense.BackgroundamplificationatRTintheabsenceofprimers,canconstituteatroublingfactorinquantitativenucleicaciddetermination,leadingtomajorinterference,particularlywhenbothsenseandantisensesequencesarepresentinthesample.SinceRTintroducedasignificantbackgrounds...
