WeroutinelyproducedsRNAbyinvitrotranscriptionofaPCRgeneratedDNAtemplatecontainingtheT7promotersequenceonbothends(I.PrimerDesigneddsRNA).ItisalsopossIBLetoproducedsRNAusingPCRgeneratedDNAtemplatescontainingeithertheT7&SP6ortheT7&T3promotersoneitherend(II.dsRNAFomClones).Thismethodislessefficient,especiallywhenworkingonalargescale. **Allworkshouldbedoneinasterile,RNasefreeenvironment,usingonlysterile,RNasefreesolutionsandmaterials,andwhilewearingglovesdoreducecontamination. I.PrimerDesigneddsRNA A.TemplateSelection TemplatescanbegeneratedbyPCRonCDNA(includingESTsfromBDGP),genomicDNA,orfirststrandRT-cDNA.MostofthedsRNAshouldcorrespondtoexonsbutdsRNAwithtwoormoreexonsinterruptedbyintronswillalsoworkwell.Wegenerallyaimfor~500bpproductsalthoughRNAiwithproductsrangingfrom150-3000bphavebeenshowntowork.Thetargetsequenceshouldnotcontaincomplete21-merhomologytoothergenesoryourdsRNAcouldbenon-specific.Onecasehasbeenseenwherethetemplatecontainedonemismatchina21-meridenticalstretchtoanothergene,andstillonlytheleveloftheintendedtargetwasreduced,whiletheclosehomologuewasunaffected.SincethetargetedregionwithinatranscriptmayinfluencethesuccessofRNAi,thesafestapproachistousedsRNAcorrespondingtothe5"or3"UTR.Itisrecommendedtocheckthevarioussequencesources(Publications,NCBIandBDGP,genomicandESTs)toconfirmtheprimarysequenceofagivengene. WesuggestusingthePrimer3website(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi)fordesigningprimers.Complementarysequencesshouldbe20-24nt,theTmrangebetween59?C-61?Candoptimizeagainstself-annealingbysettingtheMaxComplementarityto5andMax3"Complementarityto1.Afterchoosingtheprimersequence,addtheT7promotersequence(TAATACGACTCACTATAGGGAGA)tothe5"end. B.PCR Performastandard50or100µlPCRreactionusingyourselectedprimersequencestoamplifytheregionofinterest.Use1-2µlofa10µMprimerstock,100-200ngDNAastemplateandDNApolymerase.WehavesuccessfullyusedGeneChoiceTaq(PGCScientifics#62-6086-01),TaqPlus(Stratagene#600210),andHerculase(Stratagene#NC9690330)inthefollowingPCRreactionconditions: CheckthePCRresultstoensurethatyouhaveabandoftheexpectedsize.Irun4ulina1%agarosegelandusethesmallcombs.Ialsousa250bpladder. C.InvitroRNATranscription WeusetheAmbionMEGAscriptT7kit(Cat.#:1334)forthetranscriptionreaction.FollowtheAmbionMEGAscriptkitprotocolforTranscriptionReactionAssemblyanduse5µlofPCRtemplateper20µlreaction.ItisnotnecessarytopurifythePCRtemplatebeforetranscription.WeincubatethereactioninaheatblockorThermocyclerfor4hours.Followingincubation,weremovetheDNAtemplatewiththeDNase.Transcriptionandannealingoccursimultaneouslyandnoadditionalstepisrequiredtoannealthetwocomplementary.IfyouwantmoredsRNA,scaleupthereaction. ConfirmthatthedsRNAproductisthecorrectsizeusinga1%agarosegelwithTBEorTAEbufferandloadonly0.5µl. D.dsRNAPurification,Quantification,&Storage PurifyusingQiagen"sRNAeasy(#74104)orAmbion"sNucAwaySpinColumns(#10070).AmbionalsohasaMEGAclearcolumnwhichisreportedtoworkbuthasnotbeentestedinthislab.WhenusingQiagen"sRNAeasycolumns,followtheRNAcleanupprotocolandelutetwicetomaximizeyield.Also,ifyouscaleupthe20µlreactionandareusingQiagen"sRNAeasycolumns,dividethereactionandpurifyintwoormorecolumnsinordertonotoverloadasinglecolumn.Ifyouareperformingreactionsina96wellformat,purifythedsRNAinMilliporeMultiscreenPCRplates(#MANU03050).Inouropinion,theQiagenRNAeasy96wellplatesoftenperforminconsistentlyandcanbedifficulttouse. MeasuretheOD260of1:50dilution.CalculatetheconcentrationbymeasuringtheOD260of1:50-1:100dilution.ThenmultiplytheOD260bythedilutionfactorandanextinctioncoefficientof45(dsRNAConcentration=OD260xDiln.x45).Thestandardoutputof20µlreactionis80-200µg,with120µgasafrequentlyobservedvalue. ThedsRNAcanbestoredat-20oC,orat-70oCasaprecipitatewith0.1xsodiumacetateand2.5xethanol. II.dsRNAFromClones A.PCR TemplatescanbegeneratedbyPCRusingpurifiedplasmidasatemplate.TheentireinsertedsequencecanbeamplifiedusingT7&SP6orT7&T3promoterprimersdependingontheplasmid. CheckthePCRresultstoensurethatyouhaveabandoftheexpectedsize. B.InvitroRNATranscription WeusetheAmbionMEGAscriptT7,T3,&SP6kits(Cat.#:1334,1338,1330)forthetranscriptionreaction.WithT7&SP6orT7&T3promoters,eachtranscriptionreactionwilloccurinaseparatetube.FollowtheAmbionMEGAscriptkitprotocolanduse5µlofPCRtemplateper20µlreaction.ItisnotnecessarytopurifythePCRtemplatebeforetranscription. Whenthereactionsarecomplete(generallyafter2-4hours)check0.5µlofeachproductonanagarosegel. C.Purification,Quantification,&Storage PurifyeachstrandusingAmbion"sNucAwaySpinColumns.IwouldnotadviseusingtheQiagenRNAeasycolumnsduetothefactthatthetwostrandsneverannealedafterusingthem. Toanneal,combineequalmolaramountsofeachtranscriptintoonetube.Then,placethetubeinaboilingwaterbath,turntheheatoff,andleaveovernighttoanneal.Checkagainona1%agarosegel,measuretheconcentration,aliquot,andstore. III.AdditionalReferences WorbyCA,Simonson-LeffN,DixonJE.(2001)RNAinterferenceofgeneexpression(RNAi)inculturedDrosophilacells.SciSTKE(95):PL1. DixonLab-dixonlab.biochem.med.umich.edu-lookunder"Protocols"
