| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 508/528 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NucleicAcidDetection RNADetection |
| Related | LabelingCells FluorescenceImaging BiochemicalAssays |
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 |  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.
