| 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.Preparing1Xassaybuffer
Preparea1Xassaybufferbydilutingtheconcentratedbuffer20X(ComponentB)withsterile,distilled,nuclease-freewater.
2.PreparingStrandBrite™Greenworkingsolution
PrepareStrandBrite™Greenworkingsolutionbymakinga200-folddilutionoftheconcentratedDMSOsolutionin1Xassaybuffer.Forexample,add50µLofStrandBrite™Green(ComponentA)into10mLof1Xassaybuffer(fromStep1). Protecttheworkingsolutionfromlightbycoveringitwithfoilorplacingitinthedark.
Note1:Werecommendpreparingthissolutioninaplasticcontainerratherthanglass,asthedyemayadsorbtoglasssurfaces.
Note2:Forbestresults,thissolutionshouldbeusedwithinafewhoursofitspreparation.
3.PrepareserialdilutionsofRNAstandard(0to1µg/mL):
3.1 Add10μLof100μg/mLRNAstocksolution(ComponentC)to990µLofAssaybuffer(ComponentB)tohave1μg/mLRNAsolution,andthenperform1:3serialdilutionstogetapproximately1000,300,100,30,10,3,1and0ng/mL.
Note:UnusedRibosomalRNAStandard(ComponentC)shouldbedividedintosingleusealiquotsinnuclease-freeplasticvialsandstoredat-20ºC.
3.2 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:AddtheseriallydilutionsofRNAstandardsfrom1.4to1000ng/mLintowellsfromRS1toRS7induplicate.
4.RunRNAassay:
4.1 Add100μLofStrandBrite™Greenworkingsolution(fromStep2)toeachwelloftheRNAstandard,blankcontrol,andtestsamples(seeStep3)tomakethetotalRNAassayvolumeof200µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofStrandBrite™Greenworkingsolutionperwell.
4.2 Incubatethereactionatroomtemperaturefor2to5minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithaspectrofluorometeratEx/Em=490/545nm(cutoffat515nm).
Note:Tominimizephotobleaching,keepthetimeforfluorescencemeasurementconstantforallsamples.
4.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosecuvetteswithRNAstandardortestsamples.TheRNAconcentrationofthesamplearedeterminedaccordingtotheRNAstandardcurve.
| 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.
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定性ELISA只能粗略表示样本待测物含量在某个特定值以上或以下，定性ELISA通常设置阳性对照（P）、和阴性对照（N），结果分别用“阴性”和“阳性”表示。ELISA定性测定的“阴性”和“阳性”的判断标准是试剂盒所确定的阳性判断值，即cut-off值。
定量ELISA是通过一系列不同已知浓度的标准品所对应的OD值做出标准曲线，然后将样本的OD值代入标准曲线，计算出样本中待测物的含量。百特纯大分子Meretciel的定量ELISA试剂盒还可以。
1.步骤简单，45分钟内完成测定，比经典Lowry法快4倍而且更加方便。
2.灵敏度高，检测浓度下限达到25μg/ml，最小检测蛋白量达到0.5μg，待测样品体积为1-20μl 。
3.BCA法测定蛋白浓度不受绝大部分样品中的去污剂等化学物质的影响，可以兼容样品中高达5%的SDS，5%的Triton X-100，5%的Tween 20，60，80。
4.在20-2000μg/ml浓度范围内有良好的线性关系。
5.检测不同蛋白质分子的变异系数远小于考马斯亮蓝法蛋白定量。
谢谢谢
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
只要引物设计的可以在基因组上做，那么理论上是没问题的！
只需要用RT-PCR荧光定量试剂盒里面的第二个，专门用来做Real-timePCR的那些试剂就可以了！
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