AquickRNAmini-prepforNeurosporamycelialcultures

Lindgren,K.M.,A.Lichens-Park,J.L.LorosandJ.C.DunlapDept.ofBiochemistry,DartmouthMedicalSchool,Hanover,NH03756.

MostRNAisolationtechniquescurrentlyinusehavebeendevelopedfortheprocessingoflargequantitiesofmaterial.Thesetypicallyinvolvemultiplephenolextractions(Reinertetal.1981Mol.CellBiol.1:829-836)orguanADIniumisothio-cyanate/cesiumchloridegradients(Chirgwinetal.1979Biochem18:5294-5299)andcanbebothexpensiveandtimeconsuming.Often,however,needsarisewherequantitativelysmalleramountsofRNAareneededfrommanydifferentsamples,forexample,duringtimeseriesanalysesorwhenscreeningtransfor-mantsforexpressionofatransformedgene.Undersuchcircumstances,existingtech-niquesareoverlytimeconsumingandyieldmoreRNAthanisnecessary.TheavailABIlityofarapidRNAmini-prepisthusdesirable.Suchasystemhasbeendevelopedforisola-tingplantRNA(Nagyetal.1988PlantMolecularBIOLOGyManual,B4;ed.GelvinandSchilperoort,KlewerAcademicPublishing,pp.1-29),andwehaveadaptedthisprocedureforusewithNeurosporaand,potentially,otherfilamentousfungi.Below,wedescribetheuseofthisprocedurewith50mlmycelialcultures,althoughwehaveusedinwithequalsuccesswith5mlcultureswithoutscalingdowntheamountsofanyreagents.

Themethodinvolvestheuseofatriphenylmethanedye,aurintricarboxylic(ATA),toprotecttheRNA.ATAbindsirreversIBLytoRNAandisapotentinhibitorofmostnucleicacidbindingenzymes(Hallicketal.1977Nucl.AcidsRes.4:3055-3064).Thus,RNAmadewithprocedurecannotbeusedforinvitrotranscriptionortranslationorreversetranscriptionbutworksfineforRNA/DNAorRNA/RNAhybridizations.

TominimizeRNasecontamination,allglasswareisbakedat182°Cforatminimumoffourhours.Workwithglovedhands.Theprocedureisasfollows:

1.Conidiafromslants(grownin16x150mmtesttubescontaining8mlofsolidmedium)areresUSPendedin50mlofHorowitzcompletemedium(Horowitz1947J.Biol.Chem.171:255-262)andtheculturesgrownovernightwithshakingat30°C.A50mlculturetypicallyyieldsenoughRNAfor200gellanes(seebelow),and,asnoted,smallerculturevolumesmaybeused.

2.Flatmycelialpadsareeasiertogrindthanmycelialballs.Therefore,filterculturesusingaBuchnerfunnelontoWhatman#44filterpaper.Wrapflatmycelialpadsinaluminumfoilandfreezeindryice.DonotfreezeinEtOH/dryicebathbecausealcoholmightseepthroughfoil.Padscanbestoredat-70°Cforatleastthreeweeks.

3.WashamortarandpestlethoroughlywithwarmwaterandAlconox(FisherScientific);coolbyfillingwithliquidN2.Removefrozen,flatmyceliafromfoilandaddittotheliquidN2inmortar.Add~0.5gofbakedsandandgrindmycelialpadtoafinepowder.AddmoreN2asneeded.Mortarandpestleshouldbewashedaftereverysample.

4.Workingquicklybeforepowdercanthaw,pourorspoongroundmyceliainto15mlroundbottomSarstedttube(Sarstedttubes#60.540,Princeton,NJ)containing8mlofEbufferatroomtemperature.[Ebuffer:50mMTris-ClpH8.0,300mMNaCl,5mMEDTA,pH8.0,2%SDS;autoclaveandadd1mMATAand14mMß-mercaptoethanol.ATA=aurintricarboxylicacid,ammoniumsalt(Sigma#A0885,St.Louis,MO)]

5.ThawthepowderinEbufferin42°Cwaterbath,occasionallyshaking,togetSDSintosolution.Thisshouldtakeabout5minutes.

6.Add1.1mlof3MKCl,inverttomix,keeponicefor10min.Solutionshouldformsemi-solid,flocculentmassasK-SDSprecipitateforms.

7.Spinat3000g,4°Cinafixedanglerotor.Makesurecapsarescrewedontightlytopreventtubesfromcollapsing.

8.Passsupernatantthrough50micronMiracloth(Calbiochem#475855,LaJolla,CA)inafunnelintofreshSarstedttube.

9.Measurevolumeofaverage-sizedsample.Add0.5vol.8MLiCl,mixandstandat4°Covernight.

10.Spinat12000g,4°C,for15mininafixedanglerotor.Thoroughlyresuspendpelletin4mlsterilegd(glassdistilled)H2OwithpasteurPipette.

11.Extracttwicewithphenol/chloroform/isoamylalcohol(25:24:1),spinning12000g10minat4°Cinafixedanglerotor.Saveaqueous(upper)phase;addgdH2Oifvolumeislessthan2ml.

12.Add0.1vol3MNaOAcpH6.0,mixandadd2.5volEtOH,mix.Placeat-20°Covernightor-70°Cfor15min.

13.Spin12000g,10min,4°C.Washpelletwith70%EtOHanddrain.Pelletshouldbealightpinkorwhite.

14.Resuspendin0.4mlsterilegdH2Oinamicrofugetube.PrecipitatewithNaOAcandEtOHasinstep12.

15.Spin10-20mininmicrofuge.Washtwicewith70%EtOHanddrypellet.Resuspendin200µlofsterileRNase-freegdH2O.Storeat-70°Cforuptothreemonths.Spectophotometricquantificationmaybedoneatthispoint.

16.Load1µlontoaformaldehydegel.Electrophoreseovernightat20volts.Blotontonitrocellulose.ProbewithDNAfragmentofchoice.Exposetofilm.

Yieldsaretypicallyontheorderof1-2mgtotalRNAfromanovernight50mlculturearisingfromanaverageslant.Thenumberofsamplesabletobeprocessedusingthisprocedureislimitedbythenumberofspacesinacentrifugerotor.Wehavedoneasmanyas24samplesinoneday,anddoingseveraltimesthismanywouldbepossible.WehaveobservedonethidiumbromidestainedgelsthatthefluorescencefromtheRNAderivingfromthisminiprepisbrighterthanthatseenwhenthecorrespondingamountofstandard-prepRNAisused.ThismaybeduetoenhancedfluorescenceofRNAinthepresenceofATA.However,autoradiographyoftheblotsdoesnotshowanyRNAdegradationproducts(Figure1).Thisprocedurewouldprobablyworkfinewithothermethodsoftissuedisruption.ATAinhibitsmanynucleicacidbindingproteins,possiblybycompetingforbindingsites(BlumenthalandLanders1973.BBRC55:680-688).Therefore,themostcriticalfactorisgettingtheRNAincontactwithATAbeforenucleasescanbindtothenucleicacidanddegradeit.SupportedbyfederalgrantstoJ.J.L.andJ.C.D.

Fig.1.ATAmini-prepRNAprobedwithccg-1DNAfragment.TotalRNAfromaseriesoftransformantsintoBDAandfrq7Awasexaminedforthepresenceoftheccg-1genetranscript(Lorosetal.1989Science243:385-388).Eachlanecontains10µgofRNA(1/200ofthepreparation).WhilethefluorescencestainingoftheRNAextendedfromthe26Stobelowthe17SRNAbands(notshown),thehybridizationrevealedthepresenceofonlyasingleundegradedtranscriptineachlanecontainingtransformantRNAandnohybridizationtothemonkeycoscellRNAcontrol.

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