Description: -VijaiKrishnanPh.D,LouisianaStateUniversity -HARISHN.RAMANATHANPh.D.,NIDDK,NIH -RadmilaHrdlickova,Ph.D.,UTAustin -DorisBenbrook,Ph.D.,OUHSC Yes,IcertainlydidusethatsampleofPepMuteyougenerouslysentus
anditworkedsowellonHepG2cellsthatIhavesinceorderedafullsizeandamusingitexclusivelyforsiRNA! IcomparedtoRNAiMAXandRochesX-tremeGENEproducts,buttheseproductswerenotaseffectiveasPepMute. Thanksagainforthesample. Itwasfantastic! -MatthewJackson, Ph.D.,USDA
PepMutesiRNATransfectionReagent,formulatedfromsimulationofviruscellpenetratingpeptides(CPPs),isatotalnovelsiRNAdeliverytoolwhichprovidesmorethan95%silencingefficiencyat1nMsiRNAinvarietyofmammaliancells.Withourproprietarypeptidesimulationtechnology(PST),hundredsofviralCPPsweresimulated,synthesizedandscreenedforgenedeliveryefficacyinvarietyofmammaliancells(Figure1).PepMutereagentwasthenidentifiedandvalidatedasanexceptionallyefficientvectorforcondensingandtransfectingshort(under100bp)singleordoublestrandednucleicacidssuchassiRNA,miRNAmimicsandDNAoligoestowidespectrumofmammaliancells.
Figure1.AcartoonshowingPepMutesiRNATransfectionReagentwasdevelopedbyPST
Size:
-PepMuteReagent,1.0mL,sufficientfor~1,333reactionsbasedontransfecting0.5~5.0pmolsiRNAormiRNAmimicsin24-wellplate
-PepMuteTransfectionBuffer(5x),formulatedformaximaltransfectionefficiency,8.0mLat5xconcentratedstocksolutiontomake40mLofworkingsolution
Applications:
-siRNAmiRNAmimicsormRNAtransfection
-DNA/siRNAco-transfection
-DNAoligoestransfection
Storage:
Storeat4°CforPepMuteReagentandRTforPepMuteTransfectionBuffer(5x). Ifstoredproperly,theproductisstablefor12monthsorlonger.
Advantages:
-Excellentsilencingatfinal1.0nMsiRNA
-Over95%genesilencinginawidevarietyofcells
-Onetubereactionwitheasystandardprotocol
-CompatIBLewithserumandantibiotics
-ProtocolsadaptedtoHTS
-Verylowcytotoxicity
-Veryaffordable
SilencingEfficacyComparisonbetweenPepMutesiRNATransfectionReagentandLeADIngProducts
Figure2.ExcellentsilencingofendogenouslyexpressedKIF11(alsoknownasEG5)inHEK293cellswith1.0µlofPepMutereagentand0.5pmolEG5siRNAperwellof24-wellplate.KIF11(alsoknownasEG5)encodesamotorproteinthatbelongstothekinesin-likeproteinfamilyinvolvedinchromosomepositioningandbipolarspindleformationduringcellmitosis.AreductioninKIF11levelscausesmitoticarrest.PepMutereagenteffectivelydeliversEG5siRNA(final1.0nM)toHEK293cells,leadingtomorethan80%of"round-up"phenotypeofHEK293cells48hposttransfectionovernegativecontrol(final1.0nMwithshamEG5siRNA)whileleadingsiRNAtransfectinreagents,LipofectamineRNAiMAX(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.SilencingefficiencycomparisonofPepMuteTransfectionReagent(upperpanel)withDharmafect4(middlepanel)andLipofectamineRNAiMAX(RNAiMAX,lowerpanel)siRNATransfectionReagentsonA549cells.siRNAtargetingrenillaluciferaseatdifferentfinalconcentrationsrangingfrom0.5to20nMwasco-transfectedwithrenillaluciferasegene(0.5µgofpRL-CMVDNAperwell)bytheabovethreetransfectionreagentspermanufacturers"protocolsintoA549cellsgrowingona24-wellplate.Renillaluciferaseactivitywasdetermined36hafterpostco-transfectionwithrenillaluciferasedeterminationsystem(Promega).Theluminescencewasmeasuredfrom5.0µloflysateduring10sintegrationwithaluminometer(BeckmanCoulterLD400).Luciferaseactivitywasexpressedaslightunitsintegratedover10s(RLU)andnormalizedpermgofcellproteinbyusingtheBCAassay.Theerrorsbarsrepresentstandarddeviationderivedfromtriplicateexperiments.Luciferase-silencingefficiencywascalculatedrelativetountreatedcells.WhilePepMuteandDharmafect4reagentsdeliveredsignificantgenesilencingfrom1.0nMofrenillaluciferasesiRNA,LipofectamineRNAiMAXgavegoodknockdownonlyafter20nMwhileenhancedgeneexpressionatlowconcentrationofsiRNA(0.5and1.0nMrespectively)wasobserved.
Figure4.PepMuteTransfectionReagentknockeddownstableGFPexpressioninMCF7cell(upperpanel)andU2OScell(lowerpanel)byreverselytransfecting5.0and1.0nMGFPsiRNArespectively.Greenfluorescenceprotein(GFP)wasstablyexpressedinMCF7andU2OScells.siRNAtargetingGFPgene(rightpanel)andashamsiRNA(leftpanel)wereintroducedintoMCF7andU2OScellswithfinal5.0and1.0nMrespectivelybyreversetransfectionwithPepMuteTransfectionReagent.GFPgenesilencingwasmonitored48hposttransfectionbyaNikonfluorescencemicroscope.QuantitativeanalysisshowedthatGFPsiRNAat5.0and1.0nMdeliveredbyPepMutesiRNATransfectionReagentknockeddown90%and95%stablyexpressedGFPinMCF7andU2OScellsrespectively.
DataSheet&Protocol
-AStandardsiRNATransfectionProtocol
-AStandardsiRNA/DNACo-transfectionProtocol
-AReversesiRNATransfectionProtocolforHTS
Torequestafreetrialsample,pleaseCreateAnAccountwithustoenteryourshippingaddressandemailusatorder@Signagen.com
Testimonials:
IhadtriedyourproductpepMutetransfectionreagentonprimaryretinalneuronsandwassatisfiedwiththetransfectionefficiency.Willorder!
IreallyappreciateyousendingmeasampleofPepMutesiRNAreagent.ItestedDNA/siRNAco-transfectionusing293Tcellsandtheresultswerecompletelysatisfactory.Iwasabletoget95%knockdownofmytargetgeneat1.0nMsiRNAaswellasexpressionofplasmidDNAusingtherecommendedprotocol.Iwillhavetotestandseeifothercelllinesarealsoaseffective.IwilldefinitelythinkaboutmovingtousethisreagentoverDharmafectorOligofectamine.
ItriedPepMutereagentandIlikeit.Ithassohighefficiencyandnocytotoxicity.Iamgoingtouseit.Thankforintroducingittoourlab.
Tried3differentsiRNAswith100%silencingonA2780cell.Thatisamazing!Ialreadyplacedanorder.
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但是有在转染前，将细胞进行重新传代的情况，这样做的目的在于保持细胞的活性状态。
“转染前将细胞以1.5-4.5X104 cells/well 的量(具体接细胞数请参考表1）接种在孔板中，于37℃， 5%CO2 的条件下进行培养，18-24小时（ sf9细胞为3-4小时）后转染。”
其次要看下你选择单位的规模如何，上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
我刚开始做转染，悬浮细胞，分别做过表达和敲减，看了很多文献，大都没有提及转染后是用转染的这同一批细胞同时做pcr，wb，cck8，凋亡，细胞周期；还是说这次转染只做pcr或wb，再转染一次做cck8或细胞周期。剩下的功能试验均同前，转染一次做一次？我养的是悬浮细胞，转染后做cck8这些功能试验前需要离心换液吗？跪谢解答！
目标蛋白对细胞有毒性，导致细胞死亡；
转染试剂以及DNA用量信息需要优化，否则对细胞具有伤害；
细胞贴壁转染之后没有正常换液。
建议：考虑对目标蛋白进行截短构建、尝试其他细胞系统；摸索转染试剂以及DNA用量信息，如果转染试剂毒性太大，可以考虑尝试义翘转染试剂sinofection；对转染后的细胞进行换液处理，如果细胞状态感觉不够理想，可以考虑添加一些血清来帮助细胞恢复健康。
以上所有分析、建议的前提是，细胞培养、无菌操作等等都没有问题。祝顺利，加油~
转染分2种，一种是瞬时转染，即转染后让细胞表达目的蛋白后即提取蛋白，提一次蛋白，转染一次，这种方式一般不传代；
另一种转染为稳定转染，转染后加入一定选择压力进行筛选，没有转染的细胞不能存活，只留下转染的细胞，这种情况下可以筛选单个转染细胞，构建稳定表达某一特定蛋白或基因的细胞系。
各位版友求助，
我使用Hek293构建转染模型，瞬转5质粒，用lipo2000做转染体系。
转染48h，发现荧光较强的细胞都在爬片的边缘，比例十分少。是因为我添加试剂的手法不对吗。
同时也发现加入转染体系后细胞状态特别差。想问一下用lipo2000时可以用无双抗的10%FBSDMEM吗。我转染前6h现在用的是纯DMEM，不含FBS。
请各位大神帮忙
不建议你用MDCK细胞，非常难转染。
做的比较好的，一般都是上海地区的，你可以看下基尔顿生物。
