细胞状态检测试剂盒

Keywords:dualluciferase,RNA,targetgene

Ascientificmysterylitupbyfireflies--dualluciferasereportergene

DoyouwanttoverifytheeffectofmiRNAontargetgenes?--Dualluciferasereportergeneassay

DoyouwanttounderstandthetargetedinteractionbetweenmiRNAandlncRNA/circRNA?--Dualluciferasereportergeneassay

Doyouwanttoknowwhatthetranscriptionfactoristoadownstreamgene?--Dualluciferasereportergeneassay

Yes,thedualluciferasesystemissopowerful!

Dualluciferasereportergenesusuallyusefireflyluciferaseasthereportergeneandrenillaluciferaseasthereferencegene.Thereportersystemhastheadvantagesofrapiddetection,highsensitivity,widedynamicrangeandnointracellularactiveinterference.Itiswidelyusedintheresearchfieldsofgeneregulationandnon-codingRNAtargetedinteraction.Today,wewillfocusonthistopic.

Firstofall,let'stakealookatthebackgroundoffluromycin.

Luciferaseisageneraltermforanenzymeinnaturethatcanproducebiologicalfluorescence;Byoxidationofthesubstrateluciferin,fluorescenceisproduced.

FireflyluciferaseisderivedfromtheNorthAmericanfirefly.

Renillaluciferaseisderivedfromtheseakidney.

Althoughfireflyandrenillaluciferaseshavecompletelydifferentevolutionaryorigins,theirenzymestructuresandsubstraterequirementsarecompletelydifferent.FireflyluciferaseactsonbeetleluciferininthepresenceofATP,magnesium,andoxygen,whilerenillaluciferaseactsoncoelenterininthepresenceofoxygen.

Whatistheprincipleofdualluciferasetodetectthetranscriptionalregulationoftargetgenes?

Thereporterplasmidswereco-transfectedwiththecorrespondingplasmids,andthecellswerelysedafterappropriatestimulationortreatment.Afireflyfluorescencesignalisgeneratedwhenthefireflyluciferaseassayreagentisadded,representingthefireflyluciferasereportergene("experimental"reportergene).

Theabovereactionwasquenchedbytheadditionofrenillaluciferasedetectionreagent,andtherenillaluciferasefluorescencereaction(theactivityofthe"control"reportergene)wasactivatedsimultaneously,andasecondmeasurementwasperformedsimultaneously.

Theeffectsofpre-andpost-stimulationordifferentstimuliontheregulatoryelementsofinterestwerejudgedbythelevelofluciferaseactivity.

Theadvantagesofthekitareasfollows:

1.Rapiddetection:fromsamplelysistothecompletionofdetection,only10-15minutes;

2.Highsensitivity:Basedontheprincipleofchemiluminescence,luciferaseanditssubstratebioluminescencesystemcanbeverysensitiveandefficienttodetectgeneexpression.

3.Widedetectionrange:cellsampleswithdifferentexpressionlevelscanbedetected.

4.Nointerferenceofendogenousactivityincells.

Aftergoldmininginthesand,constantlylisteningtothevoiceofusers,wecollectedanumberofcommonproblemsofdualluciferasereportergenesforyou,andlaidasolidfoundationforsubsequentexperiments.

Q1:Doesthisproductrequirethesensitivityofthemicroplatereader?Doyouuseagloworaflash?

A1:Thisproductdoesnotrequirethesensitivityofthemicroplatereader,andusesaglowtype.

Q2:Afteraddingthefireflyfluorescentreagenttodetectthevalue,istherenillaluciferasedetectionreagentaddedtothewellofthefireflydetectionreagent?Ordoyouaddtherenillaluciferaseassaytoanewwellwithcelllysatesonly?

A2:Detectioninthesamewell,thatis,fireflyluciferaseactivitywasdetectedfirst,followedbyrenillaluciferaseactivity.

Q3:IfIonlywanttomeasurethelevelofrenillaluciferase,canIskiptheluciferasestepanddirectlyaddrenillaluciferaseworkingsolutionafterlysis?

A3:Yes.

Q4:Whatistheroleofrenillaluciferaseassaysubstrate?

A4:Theactionofthissubstratenotonlyinhibitedfireflyluciferaseactivity,butalsodetectedrenillaluciferaseactivity.

Q5:Doesitneedtobedoneoniceafterthesampleislysedandaddedtothestepsinthe96-wellplate?

A5:Ifitisnotnecessarytooperateonice,itisrecommendedtooperateatroomtemperature.

Q6:WhatisthedifferencebetweenLuminometerandfluorescencespectrophotometer?

A6:Thesampledetectedbythefluorescencespectrophotometercannotemitlightbyitself;thesampleneedstobeexcitedbyexcitationlightofaspecificwavelengthbeforeitcangeneratefluorescenceandbedetectedbythefluorescencespectrophotometer.ThesampledetectedbytheLuminometeritselfcanemitlightanddoesnotrequireexcitationlightforexcitation.Inotherwords,theluminometerisaninstrumentthatdetectschemiluminescence(fluorescence).Somemodelsoffluorescencespectrophotometersalsohavethefunctionofluminometer,thatis,canalsodetectchemiluminescence.Whetherthefluorometeryouareusingcanbeusedforchemiluminescencedeterminationpleasereadtheinstructionsoftheinstrumentcarefully.

Relatedproductrecommendation

ProductName

ProductNO.

Size

DualLuciferaseReporterGeneAssayKit

KTA8010

100T/1000T

SuperKine™Lipo3.0EfficientTransfectionReagent

BMU111-EN

0.5mL/1.5mL/1.5mL×5

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