ModifiedEberwine("antisense")RNAAmplificationProtocolChrisBarry,MD,PhD,PatBrownLab,4/22/99barry_c@cmgm.stanford.eduTheoptimalrangeforamplifyingfrommRNAis20-100nanograms.TheoptimalrangefortotalRNAis1-3micrograms.FirstStrandSynthesis:(Firstandsecondstrandsynthesisreactionsareperformedin0.2mLRNasefreePCRtubes.)mRNA,trehalose(Sigma#T-5251,make1.7MstockinDEPCwater)/DEPCwaterto9uL(finalconcentrationof0.6Mtrehalose/20uL)and"Eberwine"oligo-dT/T7primer(5"AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT15-3")1uL(1ug/uL).MaydrydownRNAinSpeedvactoconcentrate.Trehaloseisviscous;mixwellbypipeting.Heat65Cfor10minutesthenputonice.Add4uL5XFirstStrandBuffer(GibcoBRL,comeswithSuperscriptIIenzyme)2uL0.1MDTT(GibcoBRL,comeswithSuperscriptIIenzyme)1uLRNAsin(GibcoBRL#15518-012)1uL10mMdNTPmix(Pharmacia#27-2035-01,resUSPendinDEPCwater)1uLlinearacrylamide(0.1ug/uL,Ambion#9520orhomemade--seerecipebelow)1uLSuperscriptII(reversetranscriptase,GibcoBRL#18064-014)Thermocycle:37C5min,45C5min,then10cyclesalternatingbetween60C2minand55C2min.Placeoniceandkeepcoolwhileaddingsecondstrandcomponents.SecondStrandSynthesis:Addto1ststrandreaction:106uLDEPCwater15uL10XSecondStrandBuffer(recipebelow)3uL10mMdNTPmix(diluteinDEPCwater,Pharmacia#27-2035-01)1uLE.coliDNALigase(10U/uL,NEB#205L)4uLE.coliDNAPolymeraseI--holoenzyme(10U/uL,NEB#209L)1uLRNaseH(2U/uL,GibcoBRL#18021-071)Incubate16Cfor2hours.IfamplifyingfrommRNA,stopreactionwith10uL0.5MEDTAandincubatingat65Cfor10minutes.IfamplifyingfromtotalRNA,stopwith7.5uL1MNaOH/2mMEDTAandincubatingat65Cfor10minutes(latterdegradesribosomalandtRNAthatcaninterferewithsubsequentinvitrotranscriptionreaction).SampleExtractionandPrecipitation:Phenol:chloroform:isoamyl(25:24:1)extractonce(use0.5ml"phaselockgel"tubesfrom5"-3"Inc,#p1-257178).Addorganics(150uL)directlytoPCRtubes,beingcarefulnottospill.Mixbypipeting5-10X,thentransferslurrytoPhaselockgeltubes.Spin5minutesmaximumspeed(15kxg)atroomtemperature.TransferaqueousphasetoRNasefree1.5mlEppendorftube.Add70uL7.5Mammoniumacetate(inDEPCwater,0.2micronfiltered)toaqueousphase,then1mLabsoluteethanol(-20C).Vortex,centrifugeimmediatelyfor20minutes,maximumspeed,atroomtemperature.Itisimportanttospinrightawaysoasnottoprecipitateresidualproteinsorlowmolecularweightnucleotides(i.e.,freenucsorprimer).Washwith100uLabsoluteethanolonce,spin5minutes.Atthispoint,alarge(salt)pelletshouldbevisIBLe;ifnopelletisseenatall,suspectRNasecontamintionofreagentsordegradedstartingRNA.Removeallexcessethanol,drybrieflyatroomtemperature(notcompletelyorresuspensionwillbedifficult).Resuspendin10uLDEPCwater.(Maystophereindefinitelyat-20C.)PrepackSephadexG75spincolumns(Pharmacia#17-0050-01,makeslurrybyadding3gofpowderto50mLDEPCTE,use1ccsyringesandglasswoolplug,prespintwicefor5minutes,700xG,roomtemperature,addingresinbetweenfirstandsecondspinforfinalpackedresinvolume=0.8-1.0mL),thenpassCDNA(10uL)overcolumnfor5minutesat700xG.WhenloADIngcolumn,becarefultoputthesampleinthecenterofthematirxinordertopreventwickingofsampleontoside.Lyophilizeflowthrough(20-50uL)to16uLorlessinSpeedvac.InVitroTranscription:AmbionT7MegascriptKit(#1334),doublethestandard20uLreaction(totalvolume=40uL),37Cfor4hours.(Followmanufacturer"sinstructionsverbatim.)Phenol:chloroform:isoamylextractonce(in5"-3"phaselockgeltubes).PassoverChromaSpinTE+30column(Clontech#K1321-2).Preparecolumnswhileperformingtheorganicextraction:removetopcapthensnapoffbottom;prespin5minutesat700xg,roomtemperature.Whenloadingcolumn,becarefultoputthesampleinthecenterofthematirxinordertopreventwickingofsampleontoside.TransferflowthroughtoRnasefree1.5mLEppendorftube.Lyophilizeto13uLorless.Adjustvolumeto13uLDEPCwater,ifnecessary.Quantitateapproximateyieldby1%agaroseelectrophoresis(1uL/lane)and/orspectrophotometry(1:50or1:100dilution).ODislessaccurateduetocontaminatingprimer/freenucleotides.Gelshowswhetherthereisproductornot.Donotprocedewithlabelingifnoproductseen.ReadyforCy3/5dUTPlabelingwithrandomhexamerpriming.Labelatleast5microgramsofamplifiedRNAperreaction.Use6-8microgramsofrandomhexamersperlabelingreaction.----------------------------------------------------------------------------------10XSecondStrandBuffer:200mMTrispH6.9900mMKCl46mMMgCl21.5mMNicotineAdenineDinucleotide(Calbiochem#481915)100mM(NH4)2SO4-----------------------------------------------------------------------------------LinearAcrylamideRecipe(fromJoeDerisi,Brownlab)for375uLof10mg/mlstock:75uL5%acrylamide:3.75mgacrylamide1.5uL2MTrispH80.5uL3MNaAc0.1uL0.5MEDTA73uLwateradd1uL10%ammoniumpersulfateand0.1uLTEMEDpolymerize30minutesadd2.5volumesabsoluteetohspin5minutestopspeedaspiratesupernatantredissolvein375uL(DEPC)waterforfinalconcentrationof10ug/uLdilute1:100inDEPCwaterforuseinEberwineantisenseRNAamplificationprotocol-----------------------------------------------------------------------------------References:Luo,etal.,NatureMedicine5(1):117,1999.Carninci,etal.,PNAS95:520,1998.Eberwine,etal.,PNAS89:3010,1992.VanGelder,etal.,PNAS87:1663,1990.
