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:

1. 94^oC-3min.
2. 94^oC-45sec.
3. 57^oC-30sec.
4. 72^oC-45sec.
5. steps2-4,30x
6. 72^oC-10min.li

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-20^oC,orat-70^oCasaprecipitatewith0.1xsodiumacetateand2.5xethanol.

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"

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