Description:
PepMute™siRNATransfectionReagent,formulatedfromsimulationofviruscellpenetratingpeptides(CPPs),isatotalnovelsiRNAdeliverytoolwhichprovidesmorethan95%silencingefficiencyat1nMsiRNAinvarietyofmammaliancells.Withourproprietarypeptidesimulationtechnology(PST),hundredsofviralCPPsweresimulated,synthesizedandscreenedforgenedeliveryefficacyinvarietyofmammaliancells(Figure1).PepMute™reagentwasthenidentifiedandvalidatedasanexceptionallyefficientvectorforcondensingandtransfectingshort(under100bp)singleordoublestrandednucleicacidssuchassiRNA,miRNAmimicsandDNAoligoestowidespectrumofmammaliancells.


Figure1.AcartoonshowingPepMute™siRNATransfectionReagentwasdevelopedbyPST

Size:
-PepMute™Reagent,1.0mL,sufficientfor~1,333reactionsbasedontransfecting0.5~5.0pmolsiRNAormiRNAmimicsin24-wellplate
-PepMute™TransfectionBuffer(5x),formulatedformaximaltransfectionefficiency,8.0mLat5xconcentratedstocksolutiontomake40mLofworkingsolution

Applications:
-siRNAmiRNAmimicsormRNAtransfection
-DNA/siRNAco-transfection
-DNAoligoestransfection

Storage:
Storeat4°CforPepMute™ReagentandRTforPepMute™TransfectionBuffer(5x). Ifstoredproperly,theproductisstablefor12monthsorlonger.

Advantages:
-Excellentsilencingatfinal1.0nMsiRNA
-Over95%genesilencinginawidevarietyofcells
-Onetubereactionwitheasystandardprotocol
-CompatIBLewithserumandantibiotics
-ProtocolsadaptedtoHTS
-Verylowcytotoxicity
-Veryaffordable

SilencingEfficacyComparisonbetweenPepMute™siRNATransfectionReagentandLeADIngProducts

Figure2.ExcellentsilencingofendogenouslyexpressedKIF11(alsoknownasEG5)inHEK293cellswith1.0µlofPepMute™reagentand0.5pmolEG5siRNAperwellof24-wellplate.KIF11(alsoknownasEG5)encodesamotorproteinthatbelongstothekinesin-likeproteinfamilyinvolvedinchromosomepositioningandbipolarspindleformationduringcellmitosis.AreductioninKIF11levelscausesmitoticarrest.PepMute™reagenteffectivelydeliversEG5siRNA(final1.0nM)toHEK293cells,leadingtomorethan80%of"round-up"phenotypeofHEK293cells48hposttransfectionovernegativecontrol(final1.0nMwithshamEG5siRNA)whileleadingsiRNAtransfectinreagents,Lipofectamine™RNAiMAX(RNAiMAX,1.0nMEG5siRNA)/INTERFERin(1.0nMEG5siRNA)/Dharmafect(10.0nMEG5siRNA)andjetPRIME(20nMEG5siRNA)giveaverage37%,23%,53%and48%ball-shapedphenotyperespectivelyonHEK293cells.Thephenotypeof"rounded-up"293cellswerevisualized(upperpanel)andquantified(lowerpanel)48hposttransfectionwithaNikonmicroscope.


Figure3.SilencingefficiencycomparisonofPepMute™TransfectionReagent(upperpanel)withDharmafect4(middlepanel)andLipofectamine™RNAiMAX(RNAiMAX,lowerpanel)siRNATransfectionReagentsonA549cells.siRNAtargetingrenillaluciferaseatdifferentfinalconcentrationsrangingfrom0.5to20nMwasco-transfectedwithrenillaluciferasegene(0.5µgofpRL-CMVDNAperwell)bytheabovethreetransfectionreagentspermanufacturers"protocolsintoA549cellsgrowingona24-wellplate.Renillaluciferaseactivitywasdetermined36hafterpostco-transfectionwithrenillaluciferasedeterminationsystem(Promega).Theluminescencewasmeasuredfrom5.0µloflysateduring10sintegrationwithaluminometer(BeckmanCoulterLD400).Luciferaseactivitywasexpressedaslightunitsintegratedover10s(RLU)andnormalizedpermgofcellproteinbyusingtheBCAassay.Theerrorsbarsrepresentstandarddeviationderivedfromtriplicateexperiments.Luciferase-silencingefficiencywascalculatedrelativetountreatedcells.WhilePepMute™andDharmafect™4reagentsdeliveredsignificantgenesilencingfrom1.0nMofrenillaluciferasesiRNA,Lipofectamine™RNAiMAXgavegoodknockdownonlyafter20nMwhileenhancedgeneexpressionatlowconcentrationofsiRNA(0.5and1.0nMrespectively)wasobserved.



Figure4.PepMute™TransfectionReagentknockeddownstableGFPexpressioninMCF7cell(upperpanel)andU2OScell(lowerpanel)byreverselytransfecting5.0and1.0nMGFPsiRNArespectively.Greenfluorescenceprotein(GFP)wasstablyexpressedinMCF7andU2OScells.siRNAtargetingGFPgene(rightpanel)andashamsiRNA(leftpanel)wereintroducedintoMCF7andU2OScellswithfinal5.0and1.0nMrespectivelybyreversetransfectionwithPepMute™TransfectionReagent.GFPgenesilencingwasmonitored48hposttransfectionbyaNikonfluorescencemicroscope.QuantitativeanalysisshowedthatGFPsiRNAat5.0and1.0nMdeliveredbyPepMute™siRNATransfectionReagentknockeddown90%and95%stablyexpressedGFPinMCF7andU2OScellsrespectively.

DataSheet&Protocol
-AStandardsiRNATransfectionProtocol
-AStandardsiRNA/DNACo-transfectionProtocol
-AReversesiRNATransfectionProtocolforHTS

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Testimonials:

IhadtriedyourproductpepMutetransfectionreagentonprimaryretinalneuronsandwassatisfiedwiththetransfectionefficiency.Willorder!

-VijaiKrishnanPh.D,LouisianaStateUniversity

IreallyappreciateyousendingmeasampleofPepMutesiRNAreagent.ItestedDNA/siRNAco-transfectionusing293Tcellsandtheresultswerecompletelysatisfactory.Iwasabletoget95%knockdownofmytargetgeneat1.0nMsiRNAaswellasexpressionofplasmidDNAusingtherecommendedprotocol.Iwillhavetotestandseeifothercelllinesarealsoaseffective.IwilldefinitelythinkaboutmovingtousethisreagentoverDharmafectorOligofectamine.

-HARISHN.RAMANATHANPh.D.,NIDDK,NIH

ItriedPepMutereagentandIlikeit.Ithassohighefficiencyandnocytotoxicity.Iamgoingtouseit.Thankforintroducingittoourlab.

-RadmilaHrdlickova,Ph.D.,UTAustin

Tried3differentsiRNAswith100%silencingonA2780cell.Thatisamazing!Ialreadyplacedanorder.

-DorisBenbrook,Ph.D.,OUHSC

Yes,IcertainlydidusethatsampleofPepMuteyougenerouslysentus…anditworkedsowellonHepG2cellsthatIhavesinceorderedafullsizeandamusingitexclusivelyforsiRNA! IcomparedtoRNAiMAXandRoche’sX-tremeGENEproducts,buttheseproductswerenotaseffectiveasPepMute. Thanksagainforthesample. Itwasfantastic!

-MatthewJackson, Ph.D.,USDA