Catalog Revision A This warranty limits our liabili - PDF document

TKLs Zarrant\ OLmLts our OLabLOLt\ to repOacement of tKLs product. No otKer ZarrantLes of an\ NLnd e[press or LmpOLed LncOudLnJ ZLtKout OLmLtatLon LmpOLed ZarrantLes of mercKantabLOLt\ or fLtness for a partLcuOar purpose are provLded b\ AJLOent. AJLOent sKaOO Kave no OLabLOLt\ for an\ dLrect LndLrect consequentLaO or LncLdentaO damaJes arLsLnJ out of tKe use tKe resuOts of use or tKe LnabLOLt\ to use tKLs product. RDERING NFORMATIONwww.agilent.com ECHNICAL ERVICEStechservices@agilent.com Telephone: 800 227 9770 (option 3,4,3) www.agilent.com/en/contact-us/page MaterLaOs ProvLded ............................................................... 1StoraJe CondLtLons ............................................................... 1AddLtLonaO MaterLaOs RequLred ........................................... 1,ntroductLon .......................................................................... 2PreparLnJ tKe ReaJents ........................................................ 30% SuOfoOane ........................................................ 3 RNase-Free DNase , ............................................... 3 +LJK-SaOt WasK Buffer ........................................... 3 /oZ-SaOt WasK Buffer ............................................ 3 -MercaptoetKanoO ................................................. 3 ProtocoO ..................................................� 1;ခခခက........................... 4RNA ,soOatLon from CeOOs ....................................... 4 E[pected RNA SLJma CataOoJ #T2220] EtKanoO DLetK\Op\rocarbonate DEPC RevLsLon D0 © AJLOent TecKnoOoJLes ,nc. 200-2015 2020 2 Absolutely RNA Nanoprep Kit NTRODUCTION TKe AbsoOuteO\ RNA nanoprep NLt aOOoZs rapLd purLfLcatLon of KLJK-quaOLt\ totaO RNA from e[tremeO\ smaOO sampOes of cuOtured ceOOs ceOOs or ceOOs Karvested b\ Oaser capture mLcrodLssectLon /CTKe unLque desLJn of tKe RNA-bLndLnJ spLn cups provLded ZLtK tKAbsoOuteO\ RNA nanoprep NLt aOOoZs sampOe recover\ Ln a ver\ smaOO voOume 10 O aOOoZLnJ tKe use of tKe entLre sampOe Ln doZnstream appOLcatLons ZLtKout furtKer concentratLon steps tKat ma\ Oead to deJradatLon TKe nanoprep RNA purLfLcatLon metKod Ls outOLned Ln FLJure 1. TKe metKod empOo\s a O\sLs buffer tKat contaLns tKe cKaotropLc saOt JuanLdLne tKLoc\anate a stronJ proteLn denaturant to O\se ceOOs and prevent RNA deJradatLon b\ rLbonucOeases RNases. FoOOoZLnJ ceOO O\sLs tKe sampOe Ls transferred to a nano-spLn cup ZKere tKe RNA bLnds to a sLOLca-based fLber An optLonaO DNase dLJestLon removes contamLnatLnJ DNA tKen a serLes of ZasKes removes tKe DNase and otKer proteLns. +LJKO\ pure RNA Ls eOuted from tKe fLber matrL[ Ln a OoZ-LonLc-strenJtK buffer and Ls captured Ln a mLcrocentrLfuJe tube. TKe resuOtLnJ RNA Ls suLtabOe for use Ln a muOtLtude of moOecuOar bLoOoJ\ appOLcatLons LncOudLnJ cDNA s\ntKesLs quand quantLtatLve RT-PCR mLcroarra\ tarJet preparatLon nortKern bOottLnJ and dLfferentLaO dLspOa\. RNA Nanoprep Method Treat with DNase (optional)Wash with High Salt BufferWash twice with Low Salt Buffer Capture RNA onto fiber matrix SPIN PIN Lyse cells in Lysis BufferMix lysate with sulfolane Elute pure RNAin 10 l volume PIN PIN Absolutely RNA Nanoprep method. A bsolutely RNA Nanoprep Kit 3 REPARING THE EAGENTS 80% Sulfolane Prepare 0% v/v suOfoOane b\ dLOutLnJ 100% suOfoOane ZLtK RNaZater. PreparatLon of 5 mO of 0% suOfoOane Ls suffLcLent for processLnJ 50 RNA preparatLons from up to 0.1 mO O\sate eacK. To prepare 5 mO o0% suOfoOane add 1 mO of RNase-free Zater to 4 mO of 100% suOfoOane. Notes sulfolane is a solid at room temperature. Prior to diluting the sulfolane, melt by incubating in a 37°C waterbath until liquefied (overnight incubation is convenient for this purpose). The 80% sulfolane solution is a liquid at room temperature, and may be stored at room temperature for at least one month. If particulate matter is observed, the sulfolane solution may be filtered using a 0.2 ReconstLtute tKe O\opKLOL]ed RNase-Free DNase , b\ addLnJ 20 O of DNase ReconstLtutLon Buffer to tKe vLaO. ML[ tKe contents tKorouJKO\ to ensure tKat aOO tKe poZder Joes Lnto soOutLon. Do not Lntroduce aLr bubbOes Lnto tKe soOutLon. Store tKe r DNase Reconstitution Buffer is easily added to the vial of DNase with a syringe and needle. Gentle mixing is necessary because the DNase I is very sensitive to denaturation. High-Salt Wash Buffer Prepare 1 +LJK-SaOt WasK Buffer b\ addLnJ 16 mO of 100% etKanoO to tKe +LJK-SaOt WasK Buffer. After addLnJ tKe etKanoO marN tKe contaLner as suJJested: ffer ] 1 (Ethanol Cap tKe contaLner of 1 +LJK-SaOt WasK Buffer tLJKtO\ and store at Low-Salt Wash Buffer Prepare 1 /oZ-SaOt WasK Buffer b\ addLnJ 6 mO of 100% etKanoO to tKe After addLnJ tKe etKanoO marN tKe contaLner as suJJested: Buffer ] 1 (Ethanol . Cap tKe contaLner of 1 /oZ-SaOt WasK Buffer tLJKtO\ and store at 4 Absolutely RNA Nanoprep Kit P RNA Isolation from Cells TKe AbsoOuteO\ RNA Nanoprep .Lt Ls suLtabOe for purLfLcatLon of totaO RNA from 1–10ceOOs. For RNA preparatLon from >10ceOOs Ze offer tKe AbsoOuteO\ RNA totaO RNA mLcroprep NLt up to 5 × 10 ceOOs CataOoJ #40005 and tKe AbsoOuteO\ RNA RT-PCR mLnLprep NLt up to 10 ceOOs CataOoJ #40000. 1. Add 0. O of -ME to 100 O of /\sLs Buffer for eacK sampOe of up to Caution The Lysis Buffer contains the irritant guanidine Prepare a fresh mixture of Lysis Buffer and -ME before Cell pellets can be stored at –80°C for future processing; however, homogenizing the cells in Lysis Buffer prior to freezing the pellets is recommended to minimize RNA degradation. 2. Add 100 O of tKe /\sLs Buffer–-ME mL[ture to eacK ceOO sampOe and vorte[ or pLpet tKe sampOe repeatedO\ untLO KomoJenL]ed. Ensure that the viscosity of the lysate is low. High viscosity causes a decrease in RNA yield and an increase in DNA contamination. The viscosity can be reduced by additional vortexing, pipetting, and/or by increasing the volume of Lysis Buffer (up to 200 3. Add an equaO voOume usuaOO\ 100 O of 0% suOfoOane stored at room temperature to tKe ceOO O\sate and mL[ tKorouJKO\ b\ vorte[LnJ for It is very important to use equal volumes of 80% sulfolane and cell lysate. It is also important to vortex until the lysate and sulfolane are thoroughly mixed. 4. Transfer tKLs mL[ture to an RNA-bLndLnJ nano-spLn cup tKat Kas been seated ZLtKLn a 2-mO coOOectLon tube and snap tKe cap onto tKe top of tKe spLn cup. 5. SpLn tKe sampOe Ln a mLcrocentrLfuJe at for 60 seconds. 6. Remove and retaLn tKe spLn cup and dLscard tKe fLOtrate. Re-seat tKe spLn cup Ln tKe same 2-mO coOOectLon tube. Up to this point, the RNA has been protected from RNases by the presence of guanidine thiocyanate. A bsolutely RNA Nanoprep Kit 5 Optional DNase Treatment: TKLs procedure Ls recommended Lf DNA-free totaO RNA Ls requLred. ,f DNA removaO Ls not necessar\omLt tKe DNase treatment and proceed dLrectO\ to step . a. Add 300 O of 1× /oZ-SaOt WasK Buffer to tKe spLn cup. After cappLnJ spLn tKe sampOe Ln a mLcrocentrLfuJe at 12000 × for 60 seconds. b. Remove and retaLn tKe spLn cup and dLscard tKe fLOtrate. Re-seat tKe spLn cup Ln tKe coOOectLon tube cap tKe spLn cup and spLn tKe sampOe Ln a mLcrocentrLfuJe at 12000 × for 2 mLnutes to dr\ c. Prepare tKe DNase soOutLon b\ JentO\ mL[LnJ 2.5 O of reconstLtuted RNase-Free DNase , ZLtK 12.5 O of DNase DLJestLon Buffer for eacK sampOe. Gentle mixing is necessary because the DNase I is very sensitive to denaturation. d. Add tKe 15 O of DNase soOutLon dLrectO\ onto tKe fLber matrL[ LnsLde tKe spLn cup and cap tKe spLn cup. e. ,ncubate tKe sampOe at 3 . Add 300 O of 1× +LJK-SaOt WasK Buffer to tKe spLn cup cap tKe spLn cup and spLn tKe sampOe Ln a mLcrocentrLfuJe at 12000 × for 60 seconds. Caution The High-Salt Wash Buffer contains the irritant guanidine thiocyanate. . Remove and retaLn tKe spLn cup dLscard tKe fLOtrate and re-seat tKe spLn cup Ln tKe coOOectLon tube. Add 300 O of 1× /oZ-SaOt WasK Buffer. Cap tKe spLn cup and spLn tKe sampOe Ln a mLcrocentrLfuJe at for 60 seconds. Perform a second OoZ-saOt ZasK. Remove and retaLn tKe spLn cupdLscard tKe fLOtrate and re-seat tKe spLn cup Ln tKe coOOectLon tube. Add 300 O of 1× /oZ-SaOt WasK Buffer. Cap tKe spLn cup and spLn tKe 12000 × for 60 seconds. 11. Remove and retaLn tKe spLn cup dLscard tKe fLOtrate and re-seat tKe spLn cup Ln tKe coOOectLon tube. Cap tKe spLn cup and spLn tKe sampOe Ln a mLcrocentrLfuJe at 12000 × J for 3 mLnutes to dr\ tKe fLber matrL[12. Transfer tKe spLn cup to a fresK 2-mO coOOectLon tube. 6 Absolutely RNA Nanoprep Kit 13. Add 10 O of EOutLon Buffer dLrectO\ onto tKe fLber matrL[ LnsLde tKe spLn cup. Cap tKe spLn cup and Lncubate tKe sampOe at room temperature for 2 mLnutes. The Elution Buffer must be added directly onto the fiber ermeates the entire fiber matrix. The RNA yield may be increased by using Elution Buffer 14. SpLn tKe sampOe Ln a mLcrocentrLfuJe at 12000 × for 5 mLnutes. TKLs eOutLon step ma\ be repeated to Lncrease tKe \LeOd of totaO RNA. TKe purLfLed RNA Ls Ln tKe eOuate Ln tKe coOOectLon tube. Transfer tKe eOuate to a capped mLcrocentrLfuJe tube to store tKe RNA. TKe RNA can stored at –20°C for up to one montK or at –0°C for OonJ-term sTo quantLf\ RNA LsoOated from 10 ceOOs a KLJKO\ sensLtLve fOuorescence-based s\stem e.J. RLbo*reen RNA quantLtatLon NLt TKermo QuantLfLcatLon of RNA LsoOated from 1–10 ceOOs ma\ be acKLeved usLnJ a quantLtatLve RT-PCR QRT-PCR assa\. A t\pLcaO QRT-PCR assa\ prfor a KLJK abundance transcrLpt sucK as *APD+ and compares tKe *APD+ OeveOs Ln e[perLmentaO sampOes to tKose Ln a standard curve of totaO RNA to derLve tKe totaO RNA present Ln tKe e[perLmentaO sampOe. TKe appro[Lmate \LeOd obtaLned usLnJ dLfferent ceOO numbers as startLnJ materLaO Ls JLven Ln tKe tabOe beOoZ. Use tKe numbers presented onO\ as a JuLdeOLne e[pected resuOts ZLOO be dependent on tKe ceOO t\pe used JroZtK condLtLons and otKer factors. Cell Number Yield total RNA ~100 ng ~10 ng ~1 ng 10 0.01 ng 1 0.01 ng A bsolutely RNA Nanoprep Kit 7 Observation Suggestion RNA is degraded Use DEPC-treated or radiation-sterilized plasticware. Confirm that the cell number Confirm that 80% sulfolane and cell lysate were combined at a 1:1 ratio prior to loading the RNA-binding spin cup. Incubate the spin cup for 2 full minutes after adding the Elution least 5 minutes to collect the eluate. C prior to application to the nano-spin cup matrix. Perform the elution twice or increase the volume of Elution Buffer. Any volume between 10 and 100 l can be used. subsequent applications Concentrate the RNA under vacuum without heat. A highly viscous lysate will cause a large amount of genomic DNA to bind to the fiber matrix. Dilute the homogenate with smaller number of cells. Ensure that the spin cup is centrifuged at maximum speed 12,000 × for 2 full minutes before DNase treatment. PPENDIX SOLATING FROM ELLS ARVESTED BY ASER ICRODISSECTION TKLs protocoO can be used ZLtK CapSure transfer fLOm carrLers /CM TKermo FLsKer ScLentLfLc to LsoOate RNA from ceOOs Karvested b\ Oaser capture mLcrodLssectLon. 1. Add 0. O of -ME to 100 O of /\sLs Buffer for eacK sampOe. 2. For eacK sampOe add 100 O of tKe /\sLs Buffer–-ME mL[ture to an Eppendorf standard 0.5-mO mLcrocentrLfuJe tube. 3. Snap a CapSure transfer fLOm carrLer contaLnLnJ Oaser-captured ceOOs 4. ,nvert and vorte[ tubes for 15–30 seconds to O\se tKe ceOOs tKat Zere captured on tKe CapSure transfer fLOm carrLer. 5. Proceed ZLtK tKe protocoO Ln RNA Isolation from Cells beJLnnLnJ ZLtK 8 Absolutely RNA Nanoprep Kit PPENDIX REVENTING AMPLE ONTAMINATION RLbonucOeases are ver\ stabOe en]\mes tKat K\droO\]e RNA. RNase A can be temporarLO\ denatured under e[treme condLtLons but Lt readLO\ renatures. RNase A can tKerefore survLve autocOavLnJ and otKer standard metKods of proteLn LnactLvatLon. TKe foOOoZLnJ precautLons can KeOp prevent RNase JOoves at aOO tLmes durLnJ tKe procedures and ZKLOe KandOLnJ materLaOs and equLpment as RNases are present Ln tKe oLOs of tKe sNLn. E[ercLse care to ensure tKat aOO equLpment and suppOLes e.J. tubes etc. are free from contamLnatLnJ RNases. AvoLd usLnJ equLpment or ZorNspaces tKat Kave been e[posed to RNases. Use onO\ sterLOe tubes and mLcropLpet tLps. MLcropLpettor bores can be a source of RNase contamLnatLon sLnmaterLaO accLdentaOO\ draZn Lnto tKe pLpet or produced b\ JasNeabrasLon can faOO Lnto RNA soOutLons durLnJ pLpettLnJ. COean mLcropLpettors accordLnJ to tKe manufacturer s recommendatLons. We recommend rLnsLnJ botK tKe LnterLor and e[terLor of tKe mLcropLpet sKaft ZLtK 0% etKanoO or 0% metKanoO. Sterilizing Labware DLsposabOe sterLOe pOastLcZare Ls JeneraOO\ free of RNases. ,f sterLOe pOastLcZare Ls unavaLOabOe components sucK as mLcrocentrLfuJe tubes can be sterLOL]ed and treated ZLtK dLetK\Op\rocarbonate DEPC ZKLcK cKemLcaOO\ modLfLes and LnactLvates en]\mes accordLnJ to tKe fprotocoO: Caution DEPC is toxic and extremely reactive. Always use DEPC in a fume hood. Read and follow the manufacturer's safety 1. Add DEPC to deLonL]ed Zater to a fLnaO concentratLon of 0.1% v/v and mL[ tKorouJKO\. 2. POace tKe pOastLcZare to be treated Lnto a separate autocOavcontaLner. CarefuOO\ pour tKe DEPC-treated Zater Lnto tKe contauntLO tKe pOastLcZare Ls submerJed. 3. /eave tKe contaLner and tKe beaNer used to prepare DEPC-treated Zater Ln a fume Kood overnLJKt. A bsolutely RNA Nanoprep Kit 9 4. For dLsposaO pour tKe DEPC-treated Zater from tKe pOastLcZare Lnto anotKer contaLner ZLtK a OLd. AutocOave tKe bottOe of Zaste DEPtreated Zater and tKe contaLner ZLtK tKe pOastLcZare for at Oea30 mLnutes. AOumLnum foLO ma\ be used to cover tKe contaLner but Lt sKouOd be KandOed ZLtK JOoves and cut from an area untoucKed b\unJOoved Kands. Nondisposable Plasticware Remove RNases from nondLsposabOe pOastLcZare ZLtK a cKOoroform rLnse. Before usLnJ tKe pOastLcZare aOOoZ tKe cKOoroform to evaporate Ln a Kood or rLnse tKe pOastLcZare ZLtK DEPC-treated Zater. Electrophoresis Gel Boxes To LnactLvate RNases on eOectropKoresLs JeO bo[es treat tKe JeO bo[es ZLtK 3% v/v K\droJen pero[Lde for 10–15 mLnutes and tKen rLnse tKem ZLtK RNase-free Zater. To LnactLvate RNases on JOassZare or metaO baNe tKe JOassZare or metaO for a mLnLmum of  Kours at 10°C. Treat Zater and soOutLons e[cept tKose contaLnLnJ TrLs base ZLtK DEPC at 0.1% v/v. DurLnJ preparatLon mL[ tKe 0.1% soOutLon tKorouJKO\ Lncubate tKe soOutLon overnLJKt at room temperature and tKen autocOave Lt prLor to use. ,f a soOutLon contaLns TrLs base prepare tKe TrLs soOutLon ZLtK autocOaved DEPC-treated Zater. ,f tKe LsoOated RNA ZLOO be used for cDNA s\ntKesLs for cDNA OLconstructLon or PCR ampOLfLcatLon Lt Ls Lmportant to remove an\ resLduaO nucOeLc acLd from equLpment tKat Zas used for prevLous nucOeLc acLd LsoOatLons. 10 Absolutely RNA Nanoprep Kit EFERENCES 1. Emmert-BucN M. R. Bonner R. F. SmLtK P. D. CKuaquL R. F. =KuanJ =. et al.16 ScLence 2452: –1001. 2. CKLrJZLn -. M. Pr]\b\Oa A. E. MacDonaOd R. -. and Rutter W. -. 1 BLocKemLstr\ 124: 524–. 3. VoJeOsteLn B. and roc NatO Acad ScL U S A 62: 615–. MaterLaO Safet\ Data SKeets MSDSs are provLded onOLne at . MSDS documents are not LncOuded ZLtK product sKLpments. 11 EFERENCE ROTOCOLAdd 0.7 l of -ME to 100 1 × 10 cells l of Lysis Buffer–-ME mixture to each cell sample and vortex or pipet repeatedly until homogenized Add an equal volume of 80% sulfolane to the cell lysate and mix by vortexing for 5 seconds Transfer this mixture to a seated RNA-binding nano-spin cup; spin in a microcentrifuge at 12,000 × and discard the filtrate Optional DNase Treatment l of 1× Low-Salt Wash Buffer and spin in a microcentrifuge at 12,000 × for 60 seconds Retain the spin cup and discard the filtrate; spin in a microcentrifuge at 12,000 × for 2 minutes to dry the filter Gently mix 2.5 l of reconstituted RNase-Free DNase I with 12.5 l of DNase l of DNase solution directly onto the fiber matrix of the spin cup Incubate at 37C for 15 minutes l of 1× High-Salt Wash Buffer and spin in a microcentrifuge at 12,000 × for 60 seconds Retain the spin cup and discard the filtrate. Add 300 l of 1× Low-Salt Wash Buffer and spin in a microcentrifuge at 12,000 × for 60 seconds Retain the spin cup and discard the filtrate. Add 300 l of 1× Low-Salt Wash Buffer and spin in a microcentrifuge at 12,000 × for 60 seconds Retain the spin cup and discard the filtrate. Spin in a microcentrifuge at 12,000 × 3 minutes to dry the fiber matrix Transfer the spin cup to a fresh 2-ml collection tube l of Elution Buffer directly onto the fiber matrix and incubate for 2 minutes at room temperature; spin in a microcentrifuge at 12,000 × for 5 minutes The purified RNA is in the eluate in the collection tube. Transfer RNA to a capped microcentrifuge tube. Store at –80°C for the long term or at –20°C for the short term. Laboratory Reagent. A bsolutely RNA Nanoprep Kit ATERIALS ROVIDEDMaterials provided 35 ml -Mercaptoethanol (-ME, 14.2 M) 300 2600U DNase Reconstitution Buffer 300 1.5 ml High-Salt Wash Buffer (1.67×) 24 ml 17 ml Elution Buffer (10 mM Tris-HCl, pH 7.5) 3 ml RNA-binding nano-spin cups and caps 50 each 2-ml collection tubes (capless) 100 Sufficient reagents are provided to isolate total RNA from 50 samples of 1–10 cells each. Once opened, store at 4°C. Once reconstituted, store at –20°C. ONDITIONSUpon Receipt: -Mercaptoethanol: Once opened, store at 4°C. RNase-Free DNase I: All Other Components: Once prepared, store at room temperature. Guanidine thiocyanate in the Lysis Buffer and High-Salt Wash Buffer is an irritant. ATERIALS EQUIREDSulfolane [Sigma (Catalog #T22209)] Ethanol Diethylpyrocarbonate (DEPC) ‹ Agilent Technologies, Inc. 2007-2015, 2020 This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. RDERING NFORMATIONwww.agilent.com ECHNICAL ERVICEStechservices@agilent.com Telephone: 800 227 9770 (option 3,4,3) www.agilent.com/en/contact-us/page Absolutely RNA Nanoprep Kit Instruction ManualCatalog #400753 Revision 0 Laboratory Reagent.