RNeasy MiniHandbook - KI

1 RNeas y Min i Ha nd bookRNeasy Mini KitForpurificationof totalRNA fro m animalcells,animal tissues, ba cteria,andye as t, and forRNAcleanupRNeasy Pr ote ct MiniKitForimmed iat e sta bilizationof RNAin har vestedanimal tissuesand subsequenttot al RNApurificationRNeasy Plant Mini KitForpurificationof totalRNA fro m plants andfilamentousfun giFo urt h Edit ionJune 2012 Sample& AssayTechnologiesQI AGENS ample and AssayTec hnologiesQIA GENis thele adingpr ovider of innovativesampleandassay technologies,enablingtheisolati onanddetectionofconte ,high-qual ityproduct s and servicesensuresuccessfromsa mple to EN set s sta ndards in: Puri fi ca tionof DNA, RNA,andpr oteins Nuclei c aci d andpr otein assays mi croRNAre searchandRNAi Aut omationof sample andassay tec hnologiesOur mi ss ionis to en able youto achieveoutstandingsu in for mation.

2 Nte ntsKit Con tent s4 Storage5In ten de d Use5 Safe ty Info rm ati on6 Qual ity Con tr ol6In troduction7 Princip le andpr oc edure8De sc ri pt ionof protocol s10 Equipm ent and Reagentsto Be Suppliedby Use r13Im portant No te s16De termining th e amountof sta rting mate rial16 Handli ngandstoring sta rt ingmater ia l18Di sruptingandhomogenizingsta rti ngmater ial18 Elimina ti nggenomic DNAcontaminatio n21Pr otocolsPu rif icat io n of Tot al RNA fromAnimal Ce lls usin g Spin Tech nology23Pu rif icat io n of Tot al RNA fromAnimal Ce lls usin g Vacuum/Spi n Tec hn ol ogy29St abi li za ti on of RNAin Ha rvestedAn imal Tiss ue s34Pu rif icat io n of Tot al RNA fromAnimal Tis su es37Pu rif icat io n of Tot al RNA fromYeast43Pu rif icat io n of Tot al RNA fromPlan t Ce lls an d Tis su es andFi lam en to us Fungi50 RNAClea nup54 Trou bles hoo ti ng Gui de56Ap pendixA: Gen era l Remarkson Han dlin g RNA61Ap pendixB: Sto rage,Quantification, and Dete rmin ationof Qua li ty of RNA63Ap pendixC: Fo rma ld ehydeAgarose Ge l Ele ctr oph or es is65Ap pendixD: Opt ion al On-ColumnDNaseDigestionwith th e RNase-Free DNaseSet67Ap pendixE: DNaseDi gesti on of RNA be fore RNACle an up69Ap pendixF: Ace to ne Prec ip itatio n of Prote in fromBuf fer RLT Lysates70Ap pendixG: RT-PCR and Rea l-T ime RT-P CR71 Ord er ing Info rma ti on72 RNeasyMiniHandbook06/20123 Kit Co nte ntsRNea sy Mi ni Kit(50)(250 )Catal og no.

3 7410474 10 6Nu mber of pre ps5025 0 RNea sy MiniSpinColumns(p ink)50250 Colle ct ionTu bes(1. 5 ml )50250 Colle ct ionTu bes(2 ml)*50250 BufferRLT* 45ml220mlBufferRW1 45ml220mlBufferRP E (conc entra te)11ml65mlRNase-Free Wate r10ml50mlQuick-St ar t Pr otocol11 RNea sy Pro tect Mini Kit(50)(250 )Catal og no .7412474 12 6Nu mber of pre ps5025 0 RNAla ter RNA Stabili za tionReagent*50ml250mlRNea sy MiniSpinColumns(p ink)50250 Colle ct ionTu bes(1. 5 ml )50250 Colle ct ionTu bes(2 ml)*50250 BufferRLT* 45ml220mlBufferRW1 45ml220mlBufferRP E (conc entra te)11ml65mlRNase-Free Wate r10ml50mlQuick-St ar t Pr otocol11* Al so av ai la ge72fororderi nginformation. Contai ns a gu ani dine sa lt. Notco mpati blewi th di sinf ec forsafetyinf or matio n.

4 Be fo re usin g for thefir st ti me,add4 volumes of ethanol (96 100%)asindicatedonthebot tleto obtain awork ingsoluti on .4 RNeasyMiniHandbook06/2012 RNe asy Pl an t Mi ni Ki t(20)(50)Cat al og no .74 90374904Nu mberof preps2050 RNeasyMi ni Spi n Columns(p ink)2050QI As hr edder Spi n Columns(l il ac )2050 Col lectio n Tubes ( )2050 Col lectio n Tubes (2 ml)*2050 BufferRL T* 18ml45mlBufferRL C 18ml45mlBufferRW1 18ml45mlBufferRPE (co nc entrate)5 ml11mlRNase-FreeWater10ml10mlHandbook11* Alsoavai lable separatel y. Se e pa ge72for orderin g information . Conta insa guan idinesal t. Not co mpatib le wi th di sinf ec tantscontaini forsafetyinf or mation . Be foreusin g fo r th e fir st time, add4 vol umes of ethanol(96 100%)asindicatedonthebottle to obtain aworkingsolution.

5 St orag eTheRNeasy Mi ni Kit, RNeasy Protect MiniKit (includ ingRNAlaterRNAS tabilizationRe agent ), andRNeasyPlantMi niKitsh ould be st oreddryatroomtemperature(15 25 C)andare stableforat least 9 mont hs under th ageof RNAlaterReagent at lowerte mperat ures , redissolveth e pr eci pi ta te by heatingto 37 Cwith UseTheRNeasyMin i Kitis intende d formole cul ar biolo gyap s product is notint en dedfor thedia gnosis, pr evention,or tre atment of a duecare andattenti onshouldbeexercisedin thehandlingof l user s of QIAGEN pro duct s to ad here to theNIHguidelinesthathavebee n de ve lopedfor recombina nt DNA experiment s, or to ty Info rmationWhenworkin g wi th ch emicals,alwaysweara suitablelabcoat,disposablegloves,an d protective r more in fo rmation,ple as e co nsult theappropriate saf etydatasheets (SDSs).

6 Theseareava ila ble onl inein con ve m/safetywhe re youcanfin d, vie w, andpr in t th e SDSforea ch QI AGENkit andkit UTI ON: DO NOTad d bleachor ac idi c solutions directlyto thesam pl e preparat ion wast iocyanat e,BufferRL Ccontainsguanidinehydroc hlorid e,an d Bu fferRW1contains a small inesa lts ca n formhighlyrea ctive compoundswhencombinedwithblea ch. Ifliquidconta ini ngthesebuffers is spl it, cl ean with su itable laboratorydetergentandwater. If th e spi lt li quid con ta inspotentially in fe ctiousagents,cl eantheaffectedareafirstwith la boratorydetergentandwater , andth enwith1%(v/v) houreme rgency informati onEmergency medica l in formationinEnglish , French,andGermancanbe obtained24hours a da y fr om:PoisonInf ormationCenterMai nz,GermanyTel : +49-6131-19240 Qua lit y Con trolIn accordance withQIA GEN sIS O-certified Qual ityManagementSystem,each lotofth e RNeasyMin i Kit,RNeasyProtectMini Kit, an d RNeasyPlantMiniKitis testedagainst pr edetermined sp eci fi cation s to ensu re odu cti onTheRNeasyMini Handbookprovides pr ot ocols fo r use withthefollowingkits.

7 RNeasy Mi ni Kit forpur ificationof to talRNAfro m animalcells,animaltissues,andyea st,an d forcle anu p ofRNAfro mcrudeRNAprepsandenzymaticre actions (e. g.,DNasedigestion,proteinase digestion, RNAligation,andlabelingre action) RNeasy Prot ect Mi ni Kit forimmediate st abili zationof RNAin harvestedanimaltissue s andsu bs equentpur ificationof tot al RNARN easy Pl an t Min i Ki t forpurificationof total RNAfromplantcel ls andtissuesandfil amentousfungiTheRNeasyMiniKitcanalsobe usedto pur ifytot al RNA frombacter thiscase,westr onglyrecommendusi ngthekit in combin atio n with RNAprotect Bac teriaReagent(a vailablesepar ately),which pr ovides in vivost abiliz at ionof RNAin bacteriato ensurereliablegeneex us proto cols forstabilizingandpurifyingRNAfr omdifferentbac teriaspeciesare included inth eRNAprotectBacteriaReagentHandbook.

8 TheRNeasyMiniKit andRNAprote ctBacteriaReagentcanalsobepur ch asedtogetherastheRNeasyProte ctBacter ormation,seepages74 is alsopossible to usetheRNeasyMiniKitto purifycyt oplasmicRNAfromanimal ce ot ocol re/pr otocols/RNea syMini. are desi gnedto pu ri fy RNAfro m small amountsof y provide a fa st andsimple method fo r pr ep aring upto 100 gtotal RNApersa mple. Thepu rif iedRNAis ready forusein do wnstreamapplicationssuchas:RT -PCRan d real-timeRT -PCRD iffer entia l dis pl aycDNAsynthesisNorth ern, dot,andslotbl ot analysesPrimerextensi onPoly A+RNA sel ectionRNase/S1nuc lease prote ctionMicroarr ay sRNA-SeqTheRNeasy Ki ts allowth e para ll elprocessing dted iousmetho ds,suchasCsClstep-gradientult racent rif ugationandalcoholpre cipitati on , ormetho dsinvolvingtheuseoftoxicsubstances, suc h as phenoland /orch lo roform,ar e rep lac edbytheRNeasy in cipl e and proc edur eRNApur ific at io n usingRNeasyte chnologyTheRNeasy procedure representsa well- establ ished technologyforRNA a silica-basedmembranewith thespe edof mi cr ospin tec hnology.

9 A specializ edhigh-saltbuffersystemallowsupto 100 gof RNA longerthan 200base s to bi ndto theRNeasysilica ogicalsample s arefir st lysedan d homogenizedinthepresence ofa highlydenatur ingguanidine-thi ocyanate containingbuffer, whichimmediatelyinactivatesRNasesto en su re purificati onof intact an ol is addedto pr ovideappropriatebindingconditions, andthe sa mple is then applie d to anRNeasyMinispincolumn,where thetotal RNAbi nd s to themembrane an d co ntaminants areef fic ientlywashedaway. High-qu al ity RNAis thenelutedin 30 100 l e RNeasyprocedure,al l RNAmole cul eslongerthan200nucleotidesar oc edureprovidesan enrichment fo r mRNAsi ncemostRNAs<200nucleotides(su ,5 SrRNA,an d tRNAs,which togethercomprise15 20%of total RNA)aresel ectivelyexcluded.

10 Thesizedistributionof thepurifiedRNAis comparable to th at ob tainedby centrifugationthrougha CsClcushion,wheresmallRNAsdo notsediment otocols for purificationof smallRNAusingRNeasyKit s areavailable at www. qi thishandbook, differe nt protocols ar e pr ovided tocols dif ferprimarilyin thelysisan d homogenizationofthesampleandin theadjustmentoftheconditionsforbindingRN Ato the mpleis bou ndto themembrane,thepr otocols are similar(seeflowchart,nextpage).RNAst abi lizat io n usingRNAlatertechnologyRNAst abil iz ationis an absolute prerequisite stabili zationof RNA in bi ol ogical sample s is necessarybecause,directlyafte r ha rvestingthesamples,changesin thege ne expressionpatternoccur duetosp ecific andnonspeci fi c RNAdegradation as well as to ngesneedto be avoidedforall reliable quantit ativegeneexpressionanalyses,suchas microarrayanalyses,quanti tativeRT- PCR,suchasTaqMan andLightCyc ler te chnology,andother nu cle ic ac id- based technol MiniPro cedureRNeas y Pro te ctMin i Pro cedureRNe asy Pl antMin i Pro cedureRNeasyMiniHandbook06/20129 TheRNea sy ProtectMiniKit is suppli edwit h RNAlaterRNA StabilizationReagent.