IsolationofRNAfromDifficultTissues

TheoftenexactingprocessofisolatingintacttotalRNAfromtissuebecomesevenmoredifficultwhenprocessingcertainproblematictissues.Fibroustissuesandtissuesrichinprotein,DNAandnucleasespresentdistinctchallengesfortotalRNAisolation.Someofthedemandingtissuesrequiringmoremanipulationandfine-tuningduringtheRNAisolationprocedureareheart,brain,thymusandspleen.Hereweaddressproblemswehaveencountered,andoffertroubleshootingtechniquestohelpovercomeproblemsassociatedwithisolatingtotalRNAfromdifficulttissues.ThetipsprovidedarebasedonRNAisolationbyaguanidiniumthiocyanate/acidphenol:chloroformextractionmethod(e.g.Ambion"sToTALLYRNA™Kit).NotethatmanyofthesetechniquescanbeusedwithotherRNAisolationprotocolsaswell.

HeartandSkeletalMuscle:FibrousTissues

Forfibroustissuessuchasratandmouseheartandskeletalmuscle,themostdifficultstepintheisolationprocesscanbecompletedisruptionofallthecellswhenpreparingtissuehomogenates.Duetolowcelldensityandthepolynucleatenatureofmuscletissue,theyieldoftotalRNAistypicallylow;therefore,makingthemostofthetissueonhandiscritical.Preparationforhomogenizationshouldbecarriedoutondryice,underliquidnitrogen.PulverizingthetissueintoapowderwhilekeepingthetissuecompletelyfrozeniskeytoisolatingintacttotalRNA.LargechunksoffibroustissuearedifficulttohomogenizecompletelyandcanresultindegradedRNAandverylowyield.Unfortunately,thesignsofcomplicationoccuronlyattheendoftheisolationprocess,duringcalculationofyieldandvisualizationofthetotalRNAonagel.Therefore,careshouldbetakenduringtheinitialpreparationofthehomogenatetoensureintacttotalRNA.

BrainandPlantTissues:ProteinandLipid-richTissues

IsolatingtotalRNAfromratormousebraincanbeveryrewardingbecauseofthelargeyieldsrecoveredoncetroubleshootingtechniqueshavebeenimplemented.Brainandplanttissuesarerichinlipids,whichcancomplicatetheRNAextractionprocess,makingitdifficulttogetacleanseparationofRNA.Anobvioussignoftroubleoccursoncethebrainorplanthomogenatehasbeenextractedwithphenol:chloroform:IAA.Whiteflocculentmaterialwillmakeupmostofthevolumeoftheaqueousphaseaftercentrifugation.Thiswhitemateriallikelycontainslipidsanddoesnotformatightinterface.Toremedythesituation,addone-tenthvolumechloroform:IAA(i.e,0.1xthesumofaqueousandorganicvolumes),mixwell,andrecentrifuge.Toincreaseyield,back-extracttheorganicphaseandre-extracttheaqueousphasewithphenol:chloroform.Alternatively,remixtheaqueousandorganicphases,addmorelysissolution,effectivelydilutingtheproteinandlipids,andre-extractwithphenol:chloroform:IAA.Ithasalsobeensuggestedthatextractingaplanttissuelysatewithchloroformfirst,beforeproceedingwithRNAisolation,preventsthewhite,flocculentmaterialfromforming.

Anoptionforplanttissueinvolvestheuseofpolyvinylpyrrolidone(PVP)inthelysissteppriortotheorganicextractions.PVPcomplexeswithpolysaccharideandpolyphenolcompoundscommonlyfoundinplants.Thecomplexedmaterialiscentrifugedoutofthelysate,andthelysateisthenprocessedaccordingtoprotocol.Thissteppreventscarryoverofcontaminantsthatmayinhibitdownstreamapplications.Ambion"sPlantRNAIsolationAidisasolutionofPVPthatcanbeaddedtoplanttissuelysatesforremovalofsuchcontaminants.

RatSpleenandThymus:NucleicAcidandNucleaseRichTissues

Ratspleenandthymusarehighinnucleasesandnucleicacids.Efficienthomogenizationiscriticalforreducingtheeffectsofnucleasesfoundinthetissues.Pulverizationofthespleenandthymusintosmallpiecesondryice,underliquidnitrogen,allowsforquickhomogenizationinlysissolution,whichinactivatesnucleases.Thephenolextractionstepofthetissuelysatescanalsopresentproblems.ThehighDNAandRNAcontentofthesetissuescausesthehomogenatestobeunusuallyviscous.Extractionofsuchviscoushomogenatessometimesresultsinincompletephaseseparation.Addingmorelysissolutionand/orre-extractingwithphenol:chloroform:IAAwillhelpalleviatethisproblem.Also,multiplephenol:chloroform:IAAextractionscanbeperformedtoensurethepartitioningofDNAintotheorganicphaseduringtheacidphenolextractionsoftheRNAisolationprocedure.Ifawhiteprecipitateformsimmediatelyupontheadditionofisopropanol,post-acidphenolextraction,thisisasignthatDNAcontaminationisstillpresent.Centrifugetheprecipitate,resUSPendthepelletinnuclease-freewater,andextractwithphenol:chloroform:IAAuntilthisquick-formingwhiteprecipitatenolongeroccurs.HighyieldsofintactRNAcanbeachievedonceabundantDNAandnucleasesarebroughtundercontrol.

RNAlater™:MakingRNAIsolationFromDifficultTissuesLessDifficult

Withanyofthetissueslistedabove,oneofthechallengesistohaltRNaseactivityafterthetissueisharvested.Thisisoftenachievedbyrapidlycuttingthetissueintomanageablepieces,andplacingthepiecesintoliquidnitrogen."Snap-freezing"haltsallRNaseactivityinthetissue.Whilefrozensolid,thetissuepiecesaregroundintoapowderwithamortarandpestle.ThepowderisthenprocessedaccordingtotheRNAisolationprotocol.However,RNAcanstillbedegradedifthedissectiontakestoolongorthefrozentissuethaws.

RNAlater,anaqueous,non-toxictissueandcellstoragereagent,stABIlizesandprotectscellularRNAinintact,unfrozentissueandcellsamples.Thetissueissimplydroppedinto5volumesofRNAlater.ThereagentpermeatesthetissueimmediatelyandinactivatesRNases.RNAlater,therefore,obviatestheneedtouseliquidnitrogen,whichisfrequentlyinconvenientand,incertainsettings(e.g.fieldwork),notarealisticoptionforrapidpreservationoftissues.TissuesamplestreatedwithRNAlatercanbestoredforadayat37°C,aweekat25°C,amonthat4°C,andindefinitelyat-20°C.RNAcanbeisolatedwithstandardone-stepphenolextractionmethodsorglass-bindingmethods,aswellaswithmethodsthatuseoligod(T)selectionofmRNA.TissueissimplyremovedfromRNAlaterandprocessedlikefreshtissueintheRNAisolationlysissolution.Thereisnoneedtofreezeandgrindthetissueusingamortarandpestle,thoughfreezingandgrindingcanbedoneifdesired.(Note:RNAlatermaynotbesuitableforsomeplantandbacterialsamples.)