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AAT Bioquest/StrandBrite&trade；绿色RNA定量试剂*200X二甲基亚砜溶液*/17610/1毫升
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 500/525 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NucleicAcidDetection RNADetection |
| Related | LabelingCells FluorescenceImaging BiochemicalAssays |
1.PreparingtheStrandBrite™Greenworkingsolution:
1.1 PrepareanaqueousworkingsolutionoftheStrandBrite™Greenbymakinga200-folddilutionoftheconcentratedDMSOsolutioninTE(10mMTris-HCl,1mMEDTA,pH7.5inDEPCtreatedwater).Forexample,add50μLStrandBrite™Greento10mLTEbuffertoprepareenoughworkingsolutiontoassay100samplesina200µLfinalvolume.Protecttheworkingsolutionfromlightbycoveringitwithfoilorplacingitinthedark.
Note1:Werecommendpreparingthissolutioninaplasticcontainerratherthanglass,asthedyemayadsorbtoglasssurfaces.
Note2:Forthebestresults,thissolutionshouldbeusedwithinafewhoursofitspreparation.
2.PrepareserialdilutionsofRNAstandard(0to1µg/mL):
2.1 Preparea100μg/mLstocksolutionofRNAinDEPCtreatedwater.
2.2 Add10μLof100μg/mLRNAstocksolution(fromStep2.1)to490µLofAssaybuffer(ComponentB)tohave2μg/mLRNAsolution,andthenperform1:2serialdilutionstoget1000,500,250,125,62.5,31.3,15.6,and0ng/mL.
2.3 AddRNAstandardsandRNAcontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Table1.LayoutofRNAstandardsandtestsamplesinasolidblack96-wellmicroplate*
BL | BL | TS | TS | …. | …. |
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RS1 | RS1 | …. | …. | …. | …. |
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RS2 | RS2 |
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RS3 | RS3 |
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RS4 | RS4 |
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RS5 | RS5 |
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RS6 | RS6 |
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RS7 | RS7 |
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*Note:RS=RNAStandards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell*
RNAStandard | BlankControl | TestSample |
Serialdilutions*(100μL) | TE:100μL | 100μL |
*Note:AddtheseriallydilutionsofRNAstandardsfrom15.6to1000ng/mLintowellsfromDS1toDS7induplicate.
3.RundsDNAassay:
3.1 Add100μLofStrandBrite™Greenworkingsolution(fromStep2)toeachwelloftheRNAstandard,blankcontrol,andtestsamples(seeStep3)tomakethetotalRNAassayvolumeof200µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofStrandBrite™Greenworkingsolutionperwell.
3.2 Incubatethereactionatroomtemperaturefor5to10minutes,protectedfromlight.
3.3 MonitorthefluorescenceincreasewithaspectrofluorometeratEx/Em=490/525nm(cutoffat515nm).
Note:Tominimizephotobleachingeffects,keepthetimeforfluorescencemeasurementconstantforallsamples.
3.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosecuvetteswithRNAstandardortestsamples.TheRNAconcentrationofthesampleisdeterminedaccordingtotheRNAstandardcurve.
| References&Citations |  PrinterFriendlyVersion |
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.
