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Polyplus Transfection/INTERFERin^®/Polyplus Transfection-163147/1 Ea

Greatsilencingusingaslittleas1nMsiRNA
Over90%genesilencinginawidevarietyofcells
SuitablefortransfectionmiRNAandotheroligonucleotides
GentlemodeofactionformorerobustdataandexcellentcellviABIlity
CompatIBLewithserumandantibiotics

Specifications:

Reagent	INTERFERin^®
Moleculedelivered	siRNA,miRNA(microRNA),andotheroligonucleotides:pre-miRNA,mimicmiRNA,antimiR…
Applications	TransfectionofsiRNAandotheroligonucleotides
Celltypes	AdherentandsUSPensioncells
Numberoftransfections	1mlofINTERFERin^®issufficienttoperform500-1000transfectionsin24-wellplates.
Storage	4°C,stablefor6months(409-01)toatleastoneyear(otherpackagingsizes)whenstoredappropriately

Summary:

INTERFERin^®providesveryhighsilencingefficiencyalreadyat1nMsiRNAandcanbeusedinmoreinawidevarietyofadherentandsuspensioncells.UsinglowconcentrationofsiRNAavoidsoff-targeteffects,anditsgentlemodeofactionensuremorerobustdataandexcellentcellviability.Easytousethankstoitscompatibilitywithserumandantibiotics,INTERFERin^®isalsoperfectlysuitedfortransfectionofmiRNAandotheroligonucleotideslikepre-miRNA,mimicmiRNA,antimiR….

Orderinginformation:

|||0.1ml##409-01##|||1ml##409-10##|||5x1ml##409-50##|||

Description:

LesssiRNA,lessoff-targeteffects

SeveralpublicationsshowthattransfectinglowsiRNAconcentrationsavoidsoff-targeteffects.Indeed,theseunwantednonspecificside-effectsareobservedwhentransfectingsiRNAathighconcentrations(1,2).Hence,thereliabilityoftheexperimentaldatacanbeincreasedbyusingaslittlesiRNAaspossible.ThisiswhyINTERFERin^®hasbeenspecificallydesignedtoprovidehighsilencingefficiencyusinglowsiRNAconcentrations.
INTERFERin^®-mediateddeliveryof1nMofaspecificsiRNAshowsselectiveandhighlyefficientknockdownofgeneexpression,whileacompetitor(L2K)needsatleast10nMsiRNAtoreach50%silencingefficiency(Fig.1).

INTERFERin - Graphic comparison L2K 3 — **Fig.1:INTERFERin^®onlyrequires1nMsiRNAforefficientgenesilencing.**3LLcellsstablyexpressingfireflyluciferaseweretransfectedwithananti-LucsiRNAusingINTERFERin^®orcompetitorL2Kaccordingtothemanufacturer’srecommendation.Luciferaseexpressionwasmeasuredafter48husingaconventionalassay.NoinhibitionwasobservedwithcontrolsiRNA.

Amazingly,50%silencingisstillachievedat10pMsiRNA(Fig.2).

Fig. 2: INTERFERin<sup>®ishighlyefficientevenatextremelylowsiRNAconcentration(40%silencingevenat10pM). — **Fig.2:INTERFERin^®ishighlyefficientevenatextremelylowsiRNAconcentration.**A549-GL3Luccellsweretransfectedinthepresenceofserumwithdecreasingconcentrationsofananti-LucsiRNAusingINTERFERin^®.Luciferaseexpressionwasmeasuredafter48husingaconventionalassay.NoinhibitionwasobservedwithcontrolsiRNA.

Transfectionof1nMsiRNAtargetingendogenouslaminA/CwithINTERFERin^®drasticallyreduceslamingeneexpressiontobarelydetectablelevel(Fig.3).

INTERFERin - Gene silencing — **Fig.3:Transfectionof1nMsiRNAwithINTERFERin^®resultsinefficientandspecificgenesilencing**.CaSkicellsweretransfectedwith1nMlaminA/CsiRNAusingINTERFERin^®.After48h,laminA/Csilencingefficiencywasdeterminedbyimmunofluorescencemicroscopy.

1.Birmingham,A.etal.,(2006)NatureMethods,Vol.3,No3,199-204
2.Caffrey,D.etal.,(2011)PLoSOne.2011;6(7):e21503.Epub2011Jul5.

SuitableformiRNAtransfection

TransfectionofmicroRNA(miRNA)oligonucleotidesisincreasinglybeingusedtoanalyzeBIOLOGicaleffectsofspecificmiRNAsoncellfunction.INTERFERin^®isthereagentofchoicefordeliveringmiRNA,miRNAmimicsorpre-miRNAs.
INTERFERin^®isthelatestgenerationsiRNA&miRNAtransfectionreagent,especiallydesignedforhightransfectionefficiencyinawidevarietyofcells,resultinginhighgenesilencingorstimulationofgeneexpression.Indeed,somemiRNAarealsoknowntoinducegeneexpressionbyassociationwiththepromoterofthegeneofinterest.Forexample,INTERFERin^®-mediateddeliveryofmiR-373leadstoa3foldincreaseinE-Cadherinexpression(Fig.4).

Transfection of miR-373 with INTERFERin<sup>®induces3-foldincreaseofE-cadherinexpression — **Fig.4:TransfectionofmiR-373withINTERFERin^®enhancesE-cadherinexpression.**PC-3cellswereseededina6-wellplateandtransfectedwithmiR-373(25nM)using8µlperwellofINTERFERin^®.72haftertransfection,E-cadherinmRNAexpressionlevelwasdeterminedbyRT-qPCR.TheassaywasnormalizedwiththeHPRT-1geneexpression.

ThecationiccomponentsofINTERFERin^®requireverylittleoligonucleotideamount,resultinginagentletransfectionprocess.Inaddition,theeasyandrobustprotocolensuresreproducibleresults.OligonucleotideslikemiRNAcanbetransfectedwithsmallquantitiesofINTERFERin^®resultinginstronggenesilencingorstimulationofgeneexpression.WhatevertheconcentrationofINTERFERin^®used,theeffectstaysapproximatelythesamemakingINTERFERin^®aneconomicalreagent.Forexample,thefoldchangeusingdifferentvolumeofINTERFERin^®isalwaysbetween1.5and1.8(Fig.5).

10 nM miR-373 transfected with 6 µl INTERFERin<sup>®inPC-3cellsresultin1.8-foldincreaseinE-cadherinexpression. — **Fig.5:INTERFERin^®isalreadyefficientatverylowamounts.PC-3cellswereseededina6-wellplateandtransfectedwithmiR-373(10nM)using4,6or8µlperwellofINTERFERin^®.**72hpost-transfection,E-cadherinexpressionwasanalyzedbyWesternBlot.Theassaywasnormalizedusingβ-actinexpression.Thequantificationwasexpressedbytakingthecontrolsituationasreference.

HightransfectionefficiencyassociatedwithhighcellviabilitymakesINTERFERin^® thereagentofchoiceforgeneratingrelevantdataforscientificpublications.Indeed,thenumberofpublicationsusingINTERFERin^® formiRNAandmiRNArelatedmoleculeshasbeenexponentiallygrowingsinceitslaunch(Fig.6).

INTERFERin - Number of publications — **Fig.6:INTERFERin^®isinvolvedinanincreasingnumberofpublicationaboutmiRNAtransfection.**

Over90%genesilencing

Formanyadherentcelllinesorprimarycells,1nMsiRNAissufficienttoobtainmorethan90%genesilencing.Forsuspensioncelllines,80%silencingcanstillbereachedbyINTERFERin^®using5nMsiRNA(Table1).

INTERFERin - Cell table — **Table1:SuccessfullytransfectedcelllinesandsilencingefficienciesobtainedwithINTERFERin^®.**

Specificconditionsforvariouscelllinesareavailable inourPolyplus-transfection Database.

Excellentcellviability

Whenitcomestocellviability,INTERFERin^®outperformsothertransfectionreagents.48haftertransfectionwith1nMsiRNA,cellstransfectedwithINTERFERin^®appearhealthy,whiletoxicityisclearlyobservedwithreagentS(Fig.7).

INTERFERin - Comparison of cell morphology — **Fig.7:INTERFERin^®isextremelygentleoncells,asshowninthiscomparisonofcellmorphology48haftersiRNAtransfectionusingINTERFERin^®orcompetitorreagents**.A549-GL3Luccellsweretransfectedinthepresenceofserumwith1nMLuciferasesiRNAusingINTERFERin^®orcompetitorsSorHaccordingtothemanufacturer’sprotocol.

Easystandardprotocol

INTERFERin^®isreadytouseandtheprotocolisstraightforward.Astartingconcentrationof1nMsiRNAensuressilencingofmostgenesinmostcelltypes(Fig.8).
INTERFERin^®iscompatiblewithbothserumandantibiotics,henceavoidinganytimeconsumingwashesandmediumchanges;furThermoreINTERFERin^®canbeleftonthecellswithoutanyadverseeffects.

INTERFERin - Protocol — **Fig.8:INTERFERin^®Protocol**

TakealookatourExpertTipsinGeneticEngineeringandBiotechnologyNews.

VIDEO:siRNAtransfectionusingINTERFERin^®

Applications:

OurINTERFERin^®reagentisperfectlysuitedtoperformRNAinterferenceexperiments,whichcorrespondtothespecificinhibitionofgeneexpressionpromotedbysiRNA.siRNAsaregenespecificcomplementarydouble-strandedRNAoligonucleotidespresentinthecytoplasmofplants,wormsandmammaliancells,andcanbeefficientlytransfectedintocellswithINTERFERin^®.

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Citations:

HereisaselectionofrelevantreferencesusingINTERFERin®,moreareavailableinourPolyplus-transfectionDatabase.Carduner,L.,Picot,C.R.,Leroy-Dudal,J.,Blay,L.,Kellouche,S.,Carreiras,F.(2014).Cellcyclearrestorsurvivalsignalingthroughalphavintegrins,activationofPKCandERK1/2leadtoanoikisresistanceofovariancancerspheroids.,ExpCellRes320,329-4.Hu,B.,Castillo,E.,Harewood,L.,Ostano,P.,Reymond,A.,Dummer,R.,Raffoul,W.,Hoetzenecker,W.,Hofbauer,G.F.,Dotto,G.P.(2012).MultifocalepithelialtumorsandfieldcancerizationfromlossofmesenchymalCSLsignaling.,Cell149,1207.Lai,L.,Song,Y.,Liu,Y.,Chen,Q.,Han,Q.,Chen,W.,Pan,T.,Zhang,Y.,Cao,X.,Wang,Q.(2013).MicroRNA-92anegativelyregulatesToll-likereceptor(TLR)-triggeredinflammatoryresponseinmacrophagesbytargetingMKK4kinase.,JBiolChem288,7956.Nilsson,R.,Jain,M.,Madhusudhan,N.,Sheppard,N.G.,Strittmatter,L.,Kampf,C.,Huang,J.,Asplund,A.,Mootha,V.K.(2014).MetabolicenzymeexpressionhighlightsakeyroleforMTHFD2andthemitochondrialfolatepathwayincancer.,NatCommun5,3128.Qin,W.,Shi,Y.,Zhao,B.,Yao,C.,Jin,L.,Ma,J.,Jin,Y.(2010).MiR-24regulatesapoptosisbytargetingtheopenreADIngframe(ORF)regionofFAF1incancercells.,PLoSOne5,e9429.

Quality:

EverybatchofINTERFERin^®istestedinhouseinatransfectionassayonA549-Luccells,constitutivelyexpressingtheLuciferasegene.Thesilencingefficiencyobtainedusing1nMsiRNAandINTERFERin^®foreachbatchisindicatedontheCertificateofAnalysis.

Testimonials:

“IpreferINTERFERin[vstwocompetitors].Awesomeproductwithverycompetitiveprice.”
LatoniaTaliaferro-Smith,WinshipCancerInstituteofEmoryUniversity,UnitedStates

“Cellsseemedtobelessstressedcomparedto[competitor].IobservedlessapoptoticcellsandwillorderINTERFERinformynextexperiments.Thanksagainforthetrialsample.Bestwishes,Anja”
AnjaBremm,GoetheUniversitätFrankfurt,Germany

“IuseyourINTERFERinsiRNAreagentinprimaryhumanMacrophageswithterrificresults.”
JasonHammonds,EmoryUniversity,UnitedStates

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Polyplus Transfection/INTERFERin®/Polyplus Transfection-163147/1 Ea