Fastandreliablemini-prepRNAextractionfromNeurosporacrassa

V.Sokolovsky+,R.Kaldenhoff,M.RicciandV.E.A.Russo-MaxPlanckInstitutfürmolekulareGenetik+OnleavefromA.N.InstituteofBiochemistry,33LeninskyProspekt,Moscow117071,USSR

WehavedevelopedamethodforisolatinghighqualitytotalRNAfromN.crassamyceliathatreliablyyieldslargequantities.ItispossIBLetoextractmorethan50miniprepsatonce.

ProceduresofRNAextractionpublishedsofarfollowroughlythreedifferentapproaches,wherephenol/chloroform,guanidiniumsalts,and/orLiClisused.However,theseprotocolshavesomedisadvantages.Theyareeithertimeconsuming,oryieldlowamountsofRNA,orarenotpowerfulenoughforculturescontaininghighlevelsofnucleases.ExtractionfromstarvedculturesorplatemyceliawiththeLiClmethod(ChambersandRusso,FungalGenet.Newsl.1987.33:22-24)gavecompletelydegradedRNAasjudgedbygelelectrophoresis.IntryingtoimprovethemethodwemodifiedtheprotocolusedforChlamydomonasbyGromoffetal.(Mol.Cell.Biol.1989.9:3911-3918)whocombinedtheadvantagesofphenolextractionandLiClprecipitation.Theprocedureissuitableforrapidlygrownmycelia,starvedmycelia,myceliafromplates(4daysold)andsinglecoloniesofN.crassagrownonasorboseplate.Withthisprotocol,from20to300mg(dryweight)ofmycelium,growninliquidmediaoronplates,yieldedbetween200µgand3mgofRNAdependingonthegrowthconditionsandstrains.Fromasinglecolonywecouldisolateupto5µgtotalRNA.

Myceliumfromaliquidorsurfacegrownculturewasharvested,washed,driedthoroughlywithfilterpaperandfrozeninliquidnitrogen.SinglecoloniesincludingagarwerestampedoutusinganinvertedPasteurPipetteandfrozen.

TheprocedureofextractiongivenbelowisdescribedforEppendorftubesbutitisalsopossibletoscaleupthevolumes.Allmanipulationswereperformedatroomtemperatureifnotstatedotherwise.

-Pulverizethemyceliuminamortarwithliquidnitrogen-Transferthepowderintoamixtureof0.75mllysisbuffer(0.6MNaCl,10mMEDTA,100mMTrisHCl,pH8.0,4%SDS)and0.75mlphenol(saturatedwith0.1MTrisHCl,pH8.0)ina2mlEppendorftube.Thetubecanbefilledwithpowderedmycelium-Shakefor15-20min(EppendorfRotationsmischer3300)-Centrifugefor10min,10,000rpm-Transfertheupperphaseintoanequalvolumeofphenol(saturatedwith0.1MTrisHCl,pH8.0)andvortex-Centrifugefor10min,10,000rpm-Add0.75volumesof8MLiCltotheupperphase-Storeovernightat4°C-Vortexbrieflyandcentrifugefor10min,10,000rpm-ResUSPendthepellet,whichisnotalwaysvisible,in0.3mldoubledistilledwater,mixwith0.03ml3MNa-acetate(pH5.2)and0.75mlethanol-Storeat-20°Cfor2horat-70°Cfor30min-Centrifugefor10min,10,000rpm-Discardthesupernatantandwashtheprecipitatewith70%ethanol-DrytheRNApelletandredissolveitinDEPCtreated(diethylpolycarbonate)water-StoretheRNAsolutionat-70°C

PurityoftheRNApreparationswasassayedbyspectrophotometricmeasurements.TheA260/A230andA260/A280ratioswere2ormore,indicatingtheabsenceofanyproteinorpolysaccharidecontamination.Usually,tracesofDNApresentinRNApreparationscanbeseenintheslotsofethidiumbromidestainedformaldehydegels.Basedonthiscriterion,RNAsamplespreparedbythedescribedmethodwerefreefromcontaminatingDNA,whileclearnondegradedribosomalbandswereseeninall200extractionswemade(seeFigure1).Northernblotanalysisbyhybridizationwithseveral32P-labelledprobes(Figure2)andinvitrotranslationexperiments(Figure3)haveshownclearsignalsindicatingthepresenceofintactmRNA.

Figure1.TotalRNAs(10礸perlane)from10differentpreparations(lanes1-10)wereelectrophoresedona1.2%agarosegelcontaining2%formaldehyde.VisualizationwasdonebystainingwithethidiumbromideandirrADIationwithUV.Themajorbandscorrespondto28Sand18SrRNA,faintbandsto23S,16Sand5SrRNA.

Figure2.AutoradiogramafterNorthernblottingandhybridizationto32P-labelledCDNAinsertofarbitrarilychosencloneN6(T.Sommeretal.1989.Nucl.AcidsResearch17:5713-5723).NorthernanalysiswasdoneaccordingtoR.A.KroczekandE.Siebert1990.Analyt.Biochem.184:90-95.

Figure3.SDS-PAGE(12.5%)of35S-methionine-labelledinvitrotranslationproducts.Lane1:14C-labelledMarkerproteins,molecularwieghtsareindicatedontheleft(Mrx10(-3).Lanes2and3:RNApreparedfrommyceliagrowninrichorammoniumdepletedmediumrespectively.RNAprobescorrespondtolanes1and2inFig.1and2.Lane4:controltranslationwithoutRNA.

Acknowledgements:WethankBeateOttoforinvitrotranslationexperimentsandNiketanPanditforcriticallyreadingthemanuscript.

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