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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity*/17657/100 Tests
产品编号:17657
市  场 价:¥100060.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$5003.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity*/17657/100 Tests
商品介绍
Overview
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Ex/Em(nm)508/528
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryNucleicAcidDetection
RNADetection
RelatedLabelingCells
FluorescenceImaging
BiochemicalAssays
ThemajorchallengetoanalyzeRNAinlivecellsistheinterferencescausedbyDNA.Toaddressthesedifficulties,AATBioquesthasdevelopedtheStrandBrite™RNAGreen,anexcellentRNA-selectiveprobethatgeneratessignificantlyenhancedgreenfluorescenceuponbindingtoRNA.Ithasbeensuccessfullyusedforflowcytometricanalysisoflivecells.StrandBrite™RNAGreenreADIlygetsintolivecells.Ithastheexcitation/emissionof490/540nm.IntheDNasedigesttest,nosignificantchangeoffluorescenceintensityinfixedcellsstainedwithStrandBriteRNAGreenwasobserved.Incontrast,afterRNasedigestion,theinitialfluorescencesignaldecreasedimmediately.TheseresultsindicatethatinitialfluorescencesignalwasgeneratedfromthespecificinteractionofStrandBriteRNAGreenwithRNAincells.ShortexposureoflivecellstoantinomycinDdidcauseinhibitionofRNAsynthesisduring6hoursafterdrugremovalinadose-dependentmanner.ThesedatademonstratethatStrandBriteRNAGreenisasensitiveRNA-selectivedyeforstainingnucleolarRNAinliveandfixedcells.StrandBriteRNAGreenhaslessDNAinterferencesthanthecommonlyusedSYTO®RNASelect™dye.StrandBrite™RNAGreenisahighlyRNA-selectivefluorescentprobe.DuetoitsexcellentcellpermeABIlityandspectralproperties,ithasbeensuccessfullyusedforflowcytometricRNAanalysisandfluorescencemicroscopeinlivecells.Itcanbewellexcitedwiththe488nmbluelaserandmonitoredinFITCchannel.StrandBrite™RNAGreenprovidesavaluablemethodforidentifyingandlabelingcellswithasingleincubationstepandcandiscriminateRNAfromDNAwithbetterselectivitythanthecommonlyusedSYTO®RNASelect™.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparing1Xassaybuffer

Preparea1Xassaybufferbydilutingthe10Xassaybuffer(ComponentB)withsterile,distilled,nuclease-freewater.

2.PreparingStrandBrite™RNAGreenworkingsolution

PrepareStrandBrite™RNAGreenworkingsolutionbymakinga200-folddilutionoftheconcentratedDMSOsolutionin1Xassaybuffer(fromStep1).Forexample,add10µLofStrandBrite™RNAGreen(ComponentA)into2mLof1Xassaybuffer. Protecttheworkingsolutionfromlightbycoveringitwithfoilorplacingitinthedark.

Note1:Werecommendpreparingthissolutioninaplasticcontainerratherthanglass,asthedyemayadsorbtoglasssurfaces.

Note2:Forbestresults,thissolutionshouldbeusedwithinafewhoursofitspreparation.

3.PrepareserialdilutionsofRNAstandard(0to20µg/mL):

3.1   Add10μLof2mg/mLRNAstocksolution(ComponentC)to990µLof1Xassaybuffer(fromStep1)tohave20μg/mLRNAsolution,andthenperform1:2serialdilutionstogetapproximately20,10,5,2.5,1.25,0.625,0.313,and0µg/mL.

Note:UnusedRibosomalRNAStandard(ComponentC)shouldbedividedintosingleusealiquotsinnuclease-freeplasticvialsandstoredat≤-20ºC.

3.2   AddRNAstandardsandRNAcontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.

Table1.LayoutofRNAstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

RS1

RS1

….

….

….

….

 

 

 

 

 

 

RS2

RS2

 

 

 

 

 

 

 

 

 

 

RS3

RS3

 

 

 

 

 

 

 

 

 

 

RS4

RS4

 

 

 

 

 

 

 

 

 

 

RS5

RS5

 

 

 

 

 

 

 

 

 

 

RS6

RS6

 

 

 

 

 

 

 

 

 

 

RS7

RS7

 

 

 

 

 

 

 

 

 

 

*Note:RS=RNAStandards;BL=BlankControl;TS=TestSamples

Table2.Reagentcompositionforeachwell

RNAStandard

BlankControl

TestSample

Serialdilutions*100μL

1Xassaybuffer100μL

100μL

*Note:AddtheseriallydilutionsofRNAstandardsfrom0.3to20µg/mLintowellsfromRS1toRS7induplicate.

 

4.RunRNAassay:

4.1   Add100μLofStrandBrite™RNAGreenworkingsolution(fromStep2)toeachwelloftheRNAstandard,blankcontrol,andtestsamples(seeStep3)tomakethetotalRNAassayvolumeof200µL/well.

Note:Fora384-wellplate,add25μLsampleand25μLofStrandBrite™RNAGreenworkingsolutionperwell.

4.2   Incubatethereactionatroomtemperaturefor2to5minutes,protectedfromlight.

4.3   MonitorthefluorescenceincreasewithafluorescencemicroplatereaderatEx/Em=490/540nm(cutoffat515nm).

Note:Tominimizephotobleachingeffects,keepthetimeforfluorescencemeasurementconstantforallsamples.

References&Citations
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1.   PetersonEJ,KireevD,MoonAF,MidonM,JanzenWP,PingoudA,PedersenLC,SingletonSF.(2013)InhibitorsofStreptococcuspneumoniaesurfaceendonucleaseEndAdiscoveredbyhigh-throughputscreeningusingaPicoGreenfluorescenceassay.JBiomolScreen,18,247.

2.   ChenY,SonnaertM,RobertsSJ,LuytenFP,SchrootenJ.(2012)ValidationofaPicoGreen-basedDNAquantificationintegratedinanRNAextractionmethodfortwo-dimensionalandthree-dimensionalcellcultures.TissueEngPartCMethods,18,444.

3.   MorenoLA,CoxKL.(2010)QuantificationofdsDNAusingtheHitachiF-7000FluorescenceSpectrophotometerandPicoGreendye.JVisExp.

4.   DraganAI,Casas-FinetJR,BishopES,StrouseRJ,SchenermanMA,GeddesCD.(2010)CharacterizationofPicoGreeninteractionwithdsDNAandtheoriginofitsfluorescenceenhancementuponbinding.BiophysJ,99,3010.

5.   DraganAI,BishopES,Casas-FinetJR,StrouseRJ,SchenermanMA,GeddesCD.(2010)Metal-enhancedPicoGreenfluorescence:applicationtofastandultra-sensitivepg/mlDNAquantitation.JImmunolMethods,362,95.

6.   AbiodunOO,GbotoshoGO,AjaiyeobaEO,HappiCT,HoferS,WittlinS,SowunmiA,BrunR,OduolaAM.(2010)ComparisonofSYBRGreenI-,PicoGreen-,and[3H]-hypoxanthine-basedassaysforinvitroantimalarialscreeningofplantsfromNigerianethnomedicine.ParasitolRes,106,933.

7.   DraganAI,BishopES,Casas-FinetJR,StrouseRJ,SchenermanMA,GeddesCD.(2010)Metal-enhancedPicoGreenfluorescence:applicationfordouble-strandedDNAquantification.AnalBiochem,396,8.

8.   HoldenMJ,HaynesRJ,RabbSA,SatijaN,YangK,BlasicJR,Jr.(2009)FactorsaffectingquantificationoftotalDNAbyUVspectroscopyandPicoGreenfluorescence.JAgricFoodChem,57,7221.

9.   IkedaY,IwakiriS,YoshimoriT.(2009)DevelopmentandcharacterizationofanovelhostcellDNAassayusingultra-sensitivefluorescentnucleicacidstain"PicoGreen".JPharmBiomedAnal,49,997.

10.   RenX,XuQH.(2009)Label-freeDNAsequencedetectionwithenhancedsensitivityandselectivityusingcationicconjugatedpolymersandPicoGreen.Langmuir,25,43.


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