SuccessfulRT-PCRrequiresahighquality,intactRNAtemplate.Usethefollowingguidelinestohelppreparethistemplate: TominimizetheactivityofRNasesthatarereleasedduringcelllysis,includeRNaseinhibitorsinthelysismixorusemethodsthatsimultaneouslydisruptcellsandinactivateRNases. TakestepstoeliminateallpotentialsourcesofRNasecontaminationfromglassware,plasticware,reagents,etc. UseaproductspecificallydesignedfornucleicacidpurificationtopreparestartingtemplateRNA. UsepurifiedmRNAastemplate,ratherthantotalRNA.Startingwithpoly(A)+mRNAwillgreatlyincreasethelikelihoodofsuccessfulamplificationofraremRNAs,sincetheproportionofmRNAinatotalRNApreparationisquitelow(typically,1–5%oftotalRNAfromamammaliancell). IfusingmRNAastemplate,checktheintegrityofthemRNAbygelelectrophoresisbeforeusingitinRT-PCR.ThemRNAshouldappearasasmearbetweenapprox.500bpand8kb.MostofthemRNAshouldbebetween1.5kband2kb.