Overview

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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

DetectingandquantitatingsmallamountsofRNAisextremelyimportantforawidevarietyofmolecularBIOLOGyproceduressuchasmeasuringyieldsofinvitrotranscribedRNAandmeasuringRNAconcentrationsbeforeperformingNorthernblotanalysis,S1nucleaseassays,RNaseprotectionassays,CDNAlibrarypreparation,reversetranscriptionPCR,anddifferentialdisplayPCR.Themostcommonlyusedtechniqueformeasuringnucleicacidconcentrationisthedeterminationofabsorbanceat260nm.Themajordisadvantageoftheabsorbance-basedmethodistheinterferencescausedbyproteins,freenucleotidesandotherUVabsorbingcompounds.Theuseofsensitive,fluorescentnucleicacidstainsalleviatesthisinterferenceproblem.StrandBrite™RNAquantifyingreagentisanultrasensitivefluorescentnucleicacidstainforquantitatingRNAinsolution.StrandBrite™RNAquantifyingreagentcandetectaslittleas5ng/mLRNAwithafluorescencemicroplatereaderorfluorometer.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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*

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

Table2.Reagentcompositionforeachwell*

*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

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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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