I

Iab|e of Corterts
Package Contents and Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Storageconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Relatedproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Additionalmaterialsrecommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Noticetopurchaser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
RNAi Collection Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Vector Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
FeaturesforpRSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
pRSvectormap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
FeaturesforpGFP-V-RSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
pGFP-V-RSvectormap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
FeaturesforpRFP-C-RSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
pRFP-C-RSvectormap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
shRNAinsertdescription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Product Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Introductionofgene-specifcshRNAintomammaliancellsviatransfection . . . . 10
Introductionofgene-specifcshRNAintomammaliancellsviaretroviral
infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
Stableretroviraltransduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2
DeterminingshRNAfunctionsthroughimmunoblotting. . . . . . . . . . . . . . . . . . . . . . . . 1 2
Proteinblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
Proteindetectionwithspecifcantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
RNAiconstructvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
PlasmidDNAamplifcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
Quality Control and Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
Plasmidvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
Sequencevalidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
Transformationvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
FAQ: pRS Mammalian shRNA Expression Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8
Revision10.11TS
HuSH
TM

shRNA Plasmids (29-mer)
Appli cATi on Gui de
2
pAckAGe conTenTS And STorAGe condiTionS
pfPVRS vector
Materials Format Quantity
Gene-specifcshRNA
expressionpGFP-V-RS
vectors
Purifedandsequence-
verifedexpressionplasmids
withgene-specifcshRNA
cassettes
5ugplasmidDNAper
vial.Fouruniquecon-
structspergene.
HuSHshRNAtGFPClon-
ingVector(pGFP-V-RS)
(TR30007)
HuSH29-merNon-Effec-
tiveScrambledpGFP-V-
RS(TR30013)
Apurifedandsequence-
verifedplasmidwithout
shRNAcassetteinsert
Apurifedandsequence-
verifedplasmidcontain-
ingnon-effective29-mer
scrambledshRNAcassette
5ugplasmidDNA
5ugplasmidDNA

pRfPCRS vector
Materials Format Quantity
Gene-specifcshRNA
expressionpRFP-C-RS
vectors
Purifedandsequence-
verifedexpressionplasmids
withgene-specifcshRNA
cassettes
5ugplasmidDNAper
vial.Fouruniquecon-
structspergene.
pRFP-C-RSvector
(TR30014)
HuSH29merNon-Effec-
tive(Scrambled)pRFP-C-
RS(TR30015)
Apurifedandsequence-
verifedplasmidwithout
shRNAcassetteinsert
Apurifedandsequence-
verifedplasmidcontain-
inganon-effective29-mer
scrambledshRNAcassette
5ugplasmidDNA
5ugplasmidDNA

3
pRS vector
Materials Format Quantity
Gene-specifcshRNA
expressionpRSvectors
Purifedandsequence-
verifedexpressionplasmids
withgene-specifcshRNA
cassettes
5ugplasmidDNAper
vial.Fouruniquecon-
structspergene.
pRSVector(TR20003)
HuSH29-merNon-Ef-
fective(Scrambled)pRS
(TR30012)
Apurifedandsequence-
verifedplasmidwithout
shRNAcassetteinsert
Apurifedandsequence
verifedplasmidcontaininga
29-merscrambledcassette
5ugplasmidDNA
5ugplasmidDNA

Storae cord|t|ors
Thedriedplasmidscanbestoredat4
o
C.However,oncereconstitutedwithdH
2
O,
theplasmidsmustbestoredat-20
o
C.
Re|ated products
Positive controls:PositivecontrolshRNAexpressionvectorsareavailableforGreen
FluorescentProtein(GFP),RedFluorescentProtein(RFP)andLuciferase(Luc)inthe
pRS,pGFP-V-RS,andpRFP-C-RSvector.Theyhavebeenvalidatedforinhibitingthe
expressionoftheirrespectivetargetgenesintransienttransfectionexperimentsin
HEK293cellswithco-transfectedGFP,RFPorLuciferaseplasmids,respectively.
TR30001[HuSH29meragainstEnhancedGFP(inpRSvector)]
TR30002[HuSH29meragainstLuciferaseProtein(inpRSvector)]
TR30009[HuSH29meragainsttGFP(inpRSvector)]
TR30016[HuSH29meragainsttGFP(inpRFP-C-RSvector)]
TR30017[HuSH29meragainsttRFP(inpGFP-V-RSvector)]
PowerPrep HP Plasmid Purifcation Kits: OriGenenowoffersitsownstate-of-the-
artplasmidpurifcationtechnologytoitscustomersintheformofPowerPrepHP
MidiandMaxikits.Thesametransfection-gradeplasmidDNAavailableforourgene-
specifcproductscannowbeachievedwithPowerPrepkits.Ourplasmidpurifca-
tiontechnologyoffershigheryieldandlowerendotoxinlevelsthanmarketleaders
andutilizesion-exchangecolumnseliminatingtheuseofsyringes.Moredetailed
informationonthePowerPrepHPkitsisavailableatwww.origene.com/other/Plas-
mid_Purifcation.
RNA validation vector (TR30004):Aconvenientproducttomeasuretheeffective-

4
nessofHuSH-29orotherRNAiconstructs.Thevectorisdesignedtoincorporatea
cDNAcloneandaluciferasereportergeneasachimerictranscriptandcanbeused
toidentifythemosteffectiveknockdownconstructaswellasoptimaltransfection
conditions.Highthroughputapplicationofthisreportervectorcanbeusedtoopti-
mizeexperimentsinvolvingmultiplegenesandcelllines.Moreinformationcanbe
foundathttp://www.origene.com/rna/validation_vector.mspx.
TrueORF cDNA expression clones:OriGenehasoneofthelargestcollectionsof
cDNAclonesavailableintheworld.TrueORFcloneshavebuilt-inC-terminaltagsfor
easydetectionwithanti-tagantibodies.MoredetailedinformationontheOriGene
TrueORFcollectioncanbefoundatwww.origene.com/ORF.
Add|t|ora| ater|a|s recoerded
Transfectionreagent:Transfectionreagentsmustbeselectedandoptimizedbased
onthecelltypebeingused.Forcellsthatareinherentlydiffculttotransfect,aret-
roviralgenedeliverysystemcanbeused.OriGenesuggeststransfectionreagents
likeMegaTran1.0(OriGene),TurboFectin8.0(OriGene)orFuGENE6(Roche),
whichhavebeenshowntotransfectcommoncelltypes.
Celllineandcellculturesupplies:userpreferred
Reagentsforcelllysis:userpreferred
LB-kan(25ug/ml)LB-amp(100ug/ml)LB_Chloramphenicol(34ug/ml)liquid
culture
LB-kan(25ug/ml)orLB-amp(100ug/ml)LB_Chloramphenicol(34ug/ml)agar
plates
Reagentsandsuppliesforimmunoblots:userpreferred.OriGenehasaselection
ofantibodiesanddetectionreagentsthatareavailableatwww.origene.com/anti-
body/.
Not|ce to purcbaser
Thisproductisforresearchuseonly.Useinand/orfordiagnosticsandtherapeutics
isstrictlyprohibited.Byopeningandusingtheproduct,thepurchaseragreestothe
following:Theplasmidsmaynotbedistributed,resold,modifedforresaleorused
tomanufacturecommercialproductswithoutpriorwrittenapprovalfromOriGene
Technologies,Inc.Ifyoudonotagreetotheaboveconditions,pleasereturnthe
UNOPENEDproducttoOriGeneTechnologies,Inc.withinten(10)daysofreceiptfor
afullrefund.
rnAi collecTion overview
Asacellulardefensemechanism,hostcellsprocessdouble-strandedRNAinto
smallmoleculeswhichtargethomologousRNAsfordestruction(Hannon2002).In
mammaliancells,RNAinterference(RNAi)canbetriggeredbysiRNAsthatcause
strong,yettransientinhibitionofgeneexpressiononspecifcgenes(Elbashir2001).
ThesesiRNAscanbesynthesizedandtransfectedintomammaliancells,resultingin
effectivesuppressionofgeneexpression.Unfortunately,suchsuppressionistran-

5
sient.Bycontrast,shorthairpinRNAs(shRNA)cansuppressgeneexpressionover
aprolongedperiodbycontinuallyexpressinganRNAduplex(Brummelkamp2002;
Paddison2002).
OriGenehascreatedaretroviralsilencingplasmid(pRS)thatcontainsretroviral
longterminalrepeats(LTR)fromthemurinemoloneyleukemiavirus,thepuro-
mycinresistancegene,andaU6smallnuclearRNAgenepromoter(Hannon2002;
Elbashir2002)toeffectivelyexpresstheinsertedhairpinsequenceandtoachieve
RNAinterferenceuponintroductionintoandsubsequentprocessingbymammalian
cells.Thisplasmidhasbeenvalidatedfortransienttransfectionandfortheability
toinhibittargetedgeneexpressionwithGFP,luciferaseandHER2oncogenespecifc
hairpinDNAinserts.Moreover,ourvectorhasbeenvalidatedfordownregulationof
overexpressedGFPusingretroviralinfection.Four(4)designedandsequence-veri-
fedshRNAvectorsareofferedforeachtargetedgene.
Thegene-specifcshRNAexpressionplasmidswereconstructedusingsyntheticoli-
gonucleotidesclonedintotheBamHI/HindIIIcloningsitesofthepRSvector.Each
oftheshRNAexpressionplasmidshasa29nucleotidegene-specifcsequenceinsert
immediatelydownstreamofaU6promoterinplus(+)orientation,a7nucleotide
loop,andthe29nucleotidesequenceinreversecomplement,followedbyaTTTTTT
terminationsequence.Allinsertshavethesequencestructureshownbelow:
U6promoterGATCG--29ntsenseTCAAGAG29ntreversecomplement--
TTTTTT(termination)-GAAGCT
InsertionoftheshRNAcassetteintothepRSvectordestroystheBamHIrestriction
sitebychangingthesequencefromGGATCCtoGGATCG.
Approximately40shRNAvectorsweretestedagainsttheirtargetgenesin
co-transfectionexperimentswheretheshRNAexpressionplasmidsareintroduced
intoHEK293cellswiththeircorrespondingcDNAexpressionplasmids.Twenty-fve
tothirtypercentoftheshRNAexpressionvectorswereabletoinhibittargetgene
expressionbyatleast70%.ByprovidingfourshRNAconstructstargetingagiven
gene,weexpectthatatleastoneofthefourconstructsshouldprovide>90%gene
expressioninhibition.
TheOriGeneHuSHproductlinecontainsshRNAexpressionvectorscoveringmost
knowngenes,suchasproteinkinases,phosphatases,oncogenes,tumorsuppres-
sorgenesandothersignalingmoleculesorstructuralproteins.Foranygivengene,
four(4)independentshRNAexpressionvectorsareprovidedas5ugofpurifedand
driedplasmidpertube.Theoriginalvectorplasmid(pRSvector,TR20003;pGFP-V-
RSvectorTR30007;orpRFP-C-RSTR30014)isalsoincludedinthesamepackage.
Additionally,OriGeneprovidescustomerswithapRS,pGFP-V-RSorpRFP-C-RS
vectorcontaininganon-effective(scrambled)shRNAcassette(TR30012,TR30013
orTR30015)asaspecifcnegativecontrolforgenedownregulation.Alltheexpres-
sionshRNAcassettesaresequence-verifed.ManyoftheHuSHproductshavebeen
6
designedandannotatedtohavegoodhomologytomousesequences.Althoughour
datasuggestthatthetestedvectorswillbeabletosuppressthecorrespondinggene
expressionby70%ormore,wehavenotvalidatedtheeffectivenessofalloffered
shRNAconstructs.
PositivecontrolshRNAexpressionvectorsagainstGreenFluorescentProtein(GFP),
RedFluorescentProtein(RFP)andLuciferase(Luc)genesareavailableforpurchase
[pleaserefertopage3forcompletelist].shRNA-GFPandshRNA-Lucwerecon-
structedinthepRSandpRFP-C-RSvectorsusingthesameapproach.shRNA-RFP
wasconstructedinpGFP-V-RSvector.Eachwasshowntoinhibititsco-transfected
targetgenebyapproximately90%whenassayedafterco-transfectionintoHEK293
cellswiththeexpressionplasmidsforGFP,RFPorLuc.

Aspartofthequalitycontrolprocess,eachplasmidwastransformedinto E. Coli
andDNAwasisolatedfromasinglebacterialclone.DNAsequenceanalysiswas
performedoneachplasmidandthesequenceswerematchedtothespecifcregions
ofthetargetgenesthroughBLASTanalysis.Moreinformationisavailableonthe
OriGenewebsitehttp://www.origene.com/rnai/quality.mspx.
vecTor inforMATion
ThepRSshRNAexpressionvectorhasanumberoffeaturesallowingbothtransient
andstabletransfection,aswellasthestabledeliveryoftheshRNAexpression
cassetteintohostcellsviaareplication-defcientretrovirus.EffcacyoftheshRNA
expressionvectorsshouldbedeterminedintransienttransfectionexperiments
againstthetargetgenes.OncethesuppressingfunctionofanshRNAvectorises-
tablished,thatvectorcanbeusedtocreatestablecelllines,eitherthroughtransfec-
tionorretroviralinfection,viapuromycinselection.
TheOriGenepRSplasmidcontainsboth5and3LTRsofMoloneymurineleukemia
virus(MMLV)thatfankthepuromycinmarkerandtheU6-shRNAexpressioncas-
sette.Upontransfectionoftheplasmidsintoapackagingcellline,replication-def-
cientvirusescanbeobtainedandusedtoinfecttargetcells.Apuromycin-N-acetyl
transferasegeneislocateddownstreamoftheSV40earlypromoter,resultingin
resistancetotheantibioticpuromycin.TheshRNAexpressioncassetteconsistsofa
29nttarget-gene-specifcsequence,a7ntloop,andanother29ntreversecomple-
mentarysequence,allunderthecontrolofthehumanU6promoter.Atermination
sequence(TTTTTT)islocatedimmediatelydownstreamofthesecond29ntreverse
complementarysequencetoterminatethetranscriptionbyRNAPolIII.The29nt
gene-specifcsequencewassequence-verifedtoensureitsmatchtothetarget
gene.Adetailedmapoftheplasmidisshownonthefollowingpageandthecom-
pleteDNAsequenceoftheplasmidwithoutanshRNAexpressioncassettecanbe
foundathttp://www.origene.com/rna/vector_information.mspx.
I
features for pRS vector:
Start End Description
1 6 EcoRI
75 331 U6promoter
335 340 BamHI
379 385 HindIII
386 391 SalI
413 681 SV40promoter
7431342 Puromycin-N-acetyltransferasesequence
14412034 3LTR
23913010 pBR322ORI
31724032 Beta-lactamaseforampicillinresistance
41684639 5LTR
pRS vector ap
5 LTR
pRS shRNA Vector
5430 bp
*RC: reverse component
pBS Ori
Target Sequence
Loop
29 nt
3 LTR
U6 promoter
AMP
r
SV40 early promoter
29 nt
Target Sequence RC*
NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAA T
G
C
A
A
A
G
Puro
r
8
features for pfPVRS vector:
InadditiontoalltheusefulfeaturesofpRS,thepGFP-V-RSvectorcontainsthepCMV
driventGFPgenewhichexpressestGFPproteinconstitutivelyinmammaliancells.
Thisfeaturemakesitpossibletomonitorthetransfectioneffciency.Thebacterial
selectionmarkeriskanamycin(25ug/ml)insteadofampicillin(100ug/ml)asfound
inthepRSvector.ThedetailedvectorinformationcanbefoundontheOriGene
website(sameURLasabove).
Start End Description
1 6 EcoRI
75 331 U6promoter
335 340 BamHI
379 385 HindIII
386 391 SalI
413 604 SV40promoter
671 1270 Puromycin-N-acetyltransferasesequence
1349 1942 3LTR
2299 2918 pBR322ORI
2977 3563 PolyAsignal
3604 3667 MCS(PmeI(3604),NotI(3619,3649)MluI(3630),AsisI(3667)
3648 4380 tGFP
4467 5192 CMVpromoter
5205 6095 NeomycinphosphtransferaseforKanamycinresistance
6231 6701 5LTR
Kan
r
pGFP-V-RS shRNA Vector
7584 bp
*RC: reverse component
pBS Ori
Target Sequence
Loop
29 nt
3 LTR
U6 promoter
CMV
SV40 early promoter
29 nt
Target Sequence RC*
NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAA T
G
C
A
A
A
G
Puro
r
5 LTR
tGFP
MCS
9
features for pRfPCRS Vector:
ThepRFP-C-RSvectoralsocontainspCMVdriventRFPgenewhichexpressestRFP
proteinconstituitivelyinmammaliancells.pRFP-C-RSalsoassiststheuserin
monitoringadual-geneknockdowneffciencywhenanshRNAcassettetargeting
geneA(expressedinRFPvector)iscotransfectedalongwithanshRNAcassette
targetinggeneB(expressedinGFPvector)alongwithrespectivetargettran-
scripts.ThebacterialselectablemarkerforthevectorisChloramphenicolinstead
ofAmpicillinorKanamycin.Thedetailedvectorinformationcanbefoundonthe
OriGenewebsite(sameURLasabove).
Start End Description
1 6 EcoRI
75 331 U6promoter
335 340 BamHI
379 385 HindIII
386 391 SalI
413 604 SV40promoter
671 1270 Puromycin-N-acetyltransferasesequence
1349 1942 3LTR
2299 2918 pBR322ORI
2977 3563 PolyAsignal
3604 3667 MCS[PmeI(3604),NotI(3619,3649),MluI(3630)AsisI(3667)]
3648 4380 tRFP
44675192 CMVPromoter
5263 5922CAMrforChloramphenicolresistance
6104 6574 5LTR
Cam
r
pRFP-C-RS shRNA Vector
7452 bp
*RC: reverse component
pBS Ori
Target Sequence
Loop
29 nt
3 LTR
U6 promoter
CMV
SV40 early promoter
29 nt
Target Sequence RC*
NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAA T
G
C
A
A
A
G
Puro
r
5 LTR
tRFP
MCS
I0
sbRNA |rsert descr|pt|or
TheHuSHshRNAgene-specifcexpressioncassettesarepreparedusingsynthetic
oligonucleotides.Theseoligonucleotidesequenceswerecomputerdesignedfor
optimalsuppressionofgeneexpressionandminimaloff-targeteffects.AllshRNAse-
quencesareverifedthroughDNAsequencinganalysis.Thesequencesareprovided
totheuserontheOriGenewebsitepriortopurchase.
TheHuSHshRNAgene-specifcexpressioncassetteswereoptimizedtoincludeboth
theterminationsignalforRNAPolIIIandGCcontenttargetedat50%tofurther
improvethequalityofthegene-specifcshRNAexpressionvectors.
producT ApplicATion
|rtroduct|or of erespec|fc sbRNA |rto aa||ar ce||s v|a trarsfect|or
Add50uLofdH
2
OintoeachofthetubescontainingshRNAexpressionplasmids.
VortexthetubesbriefytoresuspendtheDNA.Theconcentrationofthissolution
is100ng/uL.
Platetheappropriateeasilytransfectedcellline(e.g.HEK293forhuman,NIH3T3
formouseorOLN-93forratshRNAvalidation)cellsat3X10
5
in2mlintoawell
ofa6-wellplate.Growthecellsovernightina5%CO
2
incubatortoachieve50%
confuence.
Inasmallsteriletube,combinethefollowingreagentsintheprescribedorder.
Theorderofreagentadditionisimportanttoachievetheoptimalresults.

Serum-freeDMEM 100uL
TurboFection8.solution 3uL
shRNAexpressionplasmidDNA 1ug

cDNAexpressionplasmidforthetargetgene 0.01ugto1.0ug
(optional,availableatOriGene)

Note: Add TurboFection 8.0 (or equivalent) directly into the serum-free media. DO
NOT let transfection reagent touch any plastic other than the pipette tip.

For Dual-gene knockdown experiment, add 50ng of each shRNA expression plas-
mids (both pGFP-V-RS vector and pRFP-C-RS vector together) with 50ng each of
target cDNAs
Mixthetubecontentsgently.DoNOTvortex!
Incubateatroomtemperaturefor15-45minutes.
AddtheDNA-TurboFectin8.0mixtothe6-wellplatedirectlywithoutremovalof
theculturemedia.Mixbygentlyswirlingtheplate.
Incubatethecellsina5%CO
2
incubatorfor48hrs.beforeharvestingforRNA
analysisand72hrs.beforeharvestingforproteinanalysis.
1.
2.
3.

II
Creation of stable cell lines without retroviral infection
TransfectthecellswiththeHuSHplasmidDNAusingyourstandardprotocolfor
transienttransfection.Aftertransfection,donotchangethemediumuntilthe
cellsarereadytobepassaged.
Passagethetransfectedcellsintoafreshvesselcontaininggrowthmedium
and0.5-1.0ug/mlpuromycin.Continuetogrowandpassagethecellsasneces-
sary,maintainingselectionpressurebykeeping0.5-1.0ug/mLpuromycininthe
growthmedium.After1-2weeks,alargenumberofthecellswillbekilledbythe
antibiotic,indicatingthattheydidnottakeuporhavelosttheplasmidwiththe
puromycinresistancecassette.Thecellsthatremaingrowinginthepuromycin
-containingmediumhaveretainedtheHuSHplasmid,whichstablyintegratesinto
thegenomeofthetargetedcells.
Selectclonalpopulationsofcellsbytransferringawell-isolatedsingleclumpof
cells(theclonalancestorandcellsdividedfromit)intoawellofa24-wellplate;
repeattoselect5-10clonalpopulations.Continuegrowingthesecellsinselec-
tionmediumfor1-2additionalpassages.Atthistime,eachwellcontainsaclonal
populationofstablytransfectedcells,whichcanbemaintainedinnormalgrowth
mediumwithouttheselectionpressureofpuromycin(althoughyoumaywishto
growthecellsunderlightpressure,0.2ug/mLpuromycin).Thesepopulations
canbeusedforexperimentsorstoredunderliquidnitrogeningrowthmedium
with10%DMSOand20%FBSforfutureuse.
|rtroduct|or of erespec|fc sbRNA |rto aa||ar ce||s v|a retrov|ra|
|rfect|or
Production of retrovirus by transient transfection of packaging cells
Platethepackagingcellsat40%confuencythedaybeforetransfection.Cells
shouldreach70-80%confuencyin24hours.
Transfectthepackagingcellsasdescribedaboveinthetransienttransfection
protocol.
Thenextday,feedthecellculturewithfreshgrowthmedia.
Onday2post-transfection,collectthemediafromtheculture,andcentrifugeat
2000xgfor5minutesorpassthrougha0.45umflter(uselowproteinbinding
flter,e.g.,celluloseacetateorpolysulfonicflter,notanitrocelluloseflter)tore-
movecelldebris.Freezethesupernatantat-80
0
Cordirectlyuseitasviralstock
forviraltiteringthroughinfectionofNIH3T3cells.
pGFP-V-RSvectorconveystGFPexpressionandpRFP-C-RSvectorconveystRFP
expressionupontransfection.HoweverthetGFPandtRFPexpressioncassettes
arelocatedoutsideoftheretroviralpackagingregion.Thevirusdoesnotpass
thetGFPortRFPintothetransducedcell.
Production of retrovirus by stable transfection of packaging cells
Platethepackagingcellsat40%confuencythedaybeforetransfection.Cells
shouldreach70-80%confuencyin24hours.
Transfectthepackagingcellsasdescribedaboveinthetransienttransfection
1.
2.
3.
1.
2.
3.
4.
5.
1.
2.
I2
protocol.
At24-36hourspost-transfection,replatethetransfectedcellsinselectionmedia
(completegrowthmediumwiththeselectionagentaddedatitsoptimal
concentration[e.g.,puromycinataconcentrationof0.5to1ug/mLforHEK293T
cells]).
Culturethecellsforoneweekusingthedrugselectionmedium.Manyofthecells
willdieduetonegativeselection,leavingonlydrug-resistantcellsalive.Select
10-20

large,healthy-lookingdrug-resistantcoloniesandtransfereachintoawell
ofanew6-wellplate.
Expandthesecoloniesintolargecultures,andcomparetheirviralyieldbyvirus
titering,ifdesired.Choosethecellswiththehighesttitertouseforvirusproduc-
tion.Otherwise,collectthemediafromtheculture,andcentrifugeat2000xg
for5minutesorpassthrougha0.45umflter(uselowproteinbindingflter,e.g.,
celluloseacetateorpolysulfonicflter,notanitrocelluloseflter)toremovecell
debris.Freezethesupernatantat-80
0
Cordirectlyuseitasviralstockforviral
titeringthroughinfectionofNIH3T3cells.
Stable retroviral transduction
Platethetargetcellsataconcentrationthatwillproduceapproximately50%
confuencyin24hours.
Addentireamountofviralstock(or,ifvirushasbeentitered,thechosencfu/mL
ofvirus)and4ug/mlpolybreneingrowthmediumdirectlyontotargetcells.Incu-
bateat37Cin5%CO
2
.
At24hourspost-infection,replacethemediumwithfreshgrowthmedium
containing0.5-1ug/mlpuromycin(ortheoptimalconcentrationasdetermined
foryourconditions).Passageasneeded,andmaintainselectionpressurefor1-2
weeks.Mostuninfectedcellsshouldbekilledbythepuromycinwithin1week.
Selectclonalpopulationsofcellsbytransferringawell-isolatedsingleclumpof
cells(theclonalancestorandcellsdividedfromit)intoawellofa24-wellplate;
repeattoselect5-10clonalpopulations.Continuegrowingthecellsinselec-
tionmediumfor1-2additionalpassages.Atthistime,eachwellcontainsaclonal
populationofstablytransfectedcells,whichcanbemaintainedinnormalgrowth
mediumwithouttheselectionpressureofpuromycin.Thesepopulationscanbe
usedforexperimentsorstoredinliquidnitrogentankingrowthmediumwith10%
DMSOand20%FBSforfutureuse.
Deter|r|r sbRNA furct|ors tbroub |urob|ott|r
Preparing cell lysates
Removetheculturemediabyaspiration.Washthecellsinthedishoncewith
ice-coldPBSandaspirateoffPBS.
Addice-coldRIPA*withfreshlyaddedproteaseinhibitorstocells(1mlper10
cmdish;0.2mlperwell/six-wellplate).Foradherentcells,rockthecellsinthe
presenceoflysisbufferinplatesinacoldroomoronicefor15minutes.For
suspensioncells,pelletthecells,thenresuspendinlysisbuffer.Transferthecell
lysissolutionintoeppendorftubes.
3.
4.
5.
1.
2.
3.
4.
1.
2.
I3
Centrifugethelysateat14,000xginapre-cooledcentrifugefor15minutes.
Immediatelytransferthesupernatanttoafreshcentrifugetubeanddiscard
thepellet.
Determinetheproteinconcentrationbyanycommerciallyavailablereagent
orkit.Atthisstep,thesamplecanbedividedintoaliquotsandstoredat80
o
C
forlong-term.
*RIPAbuffer
Stock:50mMTris-HClpH7.4,1%NP-40;0.25%Na-deoxycholate
1
,150mMNaCl,
1mMEDTA
Add fresh:1mMPMSF
2
,1ug/mlAprotinin,1ug/mlLeupeptin

1
DonotaddNa-deoxycholatewhenpreparinglysateforkinaseassays,asitmaydenaturetheproteinand
causeittoloseactivity.
2
PMSFismadeasa200mMstocksolutioninisopropanolandstoredatroomtemperature.Thevaporis
hazardous.Itisimportanttoworkwithitinachemicalhood.PMSFisnotstableinH
2
Oasithasahalf-life
ofapproximately30minutes.
Prote|r b|ott|r
Prepare3ugofcelllysatein1XLaemmlisamplebuffer
1
inavolumeof20uL(for
amini-gel,upto15ugofproteincanbeloadedperlane).Heatthesampleto70
o
C
for10min.Prepareapre-stainedproteinstandardaswell.
Runthesamplesonapre-castSDSpolyacrylamidegelwithTris-GlycineSDS
runningbufferat125Vfor90minutesuntilthedyereachesthebottomthegel.
Removethegelandsoakinproteintransferbufferfor15minutes.
PreparethePVDFmembranebypre-wettingitin100%methanol,washingonce
indH
2
Ofor5minandequilibratingitintheproteintransferbufferfor10minutes.
Assembletheelectroblottingcassetteandplacetheelectrodesintheblotting
unit,accordingtothemanufacturesinstructions.
TransferinTris-Glycinetransferbufferat25V(100mA)for1.5hours.
Followingtransfer,removethemembranefromtheblottingcassetteandmarkthe
orientationofthegelwithapencil.RinsebriefywithPBSandtrimthemem-
brane.Themembranemaybestoredat4
o
Cforseveralweeks.However,oncethe
membraneisdried,itneedstobewettedbymethanolfollowedbyPBS.
1
2XLaemmlisamplebuffer:62.5mMTris-HCl,pH6.8,2%SDS,25%glycerol,0.01%BromophenolBlue
Prote|r detect|or W|tb spec|fc art|bod|es
WashPVDFmembranewithTBST
2
oncefor5min.atroomtemperature.
Blocknon-specifcbindingonthemembranewithfreshlyprepared5%nonfat
driedmilkfor1houronashakingplatformatroomtemperature.
Washthreetimesfor5minuteseachwithTBST.
IncubatethemembranewithaspecifcprimaryantibodydilutedinTBSTand5%
BSAatthemanufacturersrecommendeddilutionwithgentleagitationat4
o
C
overnightorforseveralhoursatRT.
Washthreetimesfor5mineachwithTBST.
IncubatewithHRP-conjugatedsecondaryantibodyat1:20,000(ormanufacturers
3.
4.
1.
2.
3.
4.
5.
6.
1.
2.
3.
4.
5.
6.
I4
recommendeddilution)inTBST-5%BSAfor1houratroomtemperature.
Washthreetimesagainfor5minuteseachwithTBST.
Fordetection,usetheenhancedchemiluminescencereagentfromOriGene(West-
ernBlottingLuminolReagent(TA10006))orothercommerciallyavailabledetec-
tionsystemandprepareaccordingtothemanufacturersdirections.
Laythemembraneonaplasticsurfacewiththeproteinsideup.Addthemixed
detectionsolutiontothemembrane.Incubatefor3minutes.Removetheexcess
solutionandcoverthemembranewithtransparentplastic.
PlacethewrappedblotwithproteinsideupinanX-rayflmcassette.Placea
sheetofX-rayautoradiographyflmonthetopofthemembrane.Closethecas-
settefor1min.Removetheflmfordevelopment.Addadditionalflmsifneeded
forlongerorshorterexposures.
2
TBST:10mMTris-HCl,pH8.0,150mMNaCl,0.05%Tween20.
RNA| corstruct va||dat|or (0pt|ora|)
ThemostuptodateprotocolforthisprocesscanbefoundontheOriGenewebsite
athttp://www.origene.com/assets/Documents/HuSH/ValidationVectorProtocol.pdf
P|as|d DNA ap||fcat|or (0pt|ora|)
For pRS based HuSH vectors, the E. coli selection marker is ampicillin (100 ug/mL).
For pGFP-V-RS based HuSH vectors, the E. coli selection marker is kanamycin (25
ug/mL). For pRFP-C-RS based HuSH vectors, the E. coli selection marker is chloram-
phenicol (34 ug/ml)
Ifdesired,customerscanfrstamplifytheshRNAvectors.Add50uLofdH
2
Ointo
eachtube.VortexthetubesbriefytoresuspendtheDNA.Pipette1uLofthissolu-
tiontoanothertubeandadd99uLdH
2
O.TheconcentrationoftheDNAsolution
shouldbearound1ng/uL.Theplasmidsolutionshouldbestoredat20
o
C.
ThawtransformationcompetentE. Coli cells(standardlaboratoryDH5alpha)on
ice.Performtransformationwith1-2uLofthedilutedshRNAplasmid.Plateout
thetransformantsonLB-Kan,LB-AmporLB-Chloramphenicolplatesandincubate
overnightat37
o
C,untilcoloniesappear.
Thefollowingday,inoculatesinglebacterialcoloniesinto5mlofLB-Kan,LB-
ChloramphenicolorLB-Ampmediaandgrowthemovernight.
PurifytheDNAplasmidsfromthecultureusingaminiprepDNAisolationkit.
ResuspendtheDNAin50uLofTEsolutionanddeterminetheconcentrationof
thesamples.Storethesolutionat20
o
C.
QuAliTy conTrol And QuAliTy ASSurAnce
P|as|d va||dat|or
AllplasmidproductswithshRNAexpressioncassetteshavebeenisolatedfromsingle
colonies.Thepurifedplasmidswereexaminedonanagarosegeltoensurethepres-
enceoftheplasmidsandtoverifyquantity.
7.
8.
9.
10.
1.
2.
3.
4.
5.
I5
Sequerce va||dat|or
Thefnalproductshavebeenre-sequencedforconfrmation.
Irarsforat|or va||dat|or
ThedriedplasmidswereresuspendedindH
2
O,thenusedfortransforming E. Coli
cells.Theeffciencyoftransformationisidenticaltothatbeforedrying.
MoreQualityControlinformationisavailableattheOriGeneweb-sitehttp://www.
origene.com/rna/quality.mspx.
fAQ: prS MAMMAliAn SHrnA expreSSion plASMidS
What are the differences between the pRS, pGFP-V-RS and pRFP-C-RS ex-
pression plasmids?
Answer:ThepGFP-V-RSplasmidcontainstGFPdrivenbyaCMVpromoter.The
pRFP-C-RSplasmidcontainstRFPdrivenbyaCMVpromoter.Bothplasmids
provideaneasywaytoidentifycellsthathavebeentransfected.ThepGFP-V-RS
vectoriskanamycinresistant,pRFP-C-RSvectorischloramphenicolresistantwhile
thepRSvectorisampicillinresistant.
I am doing retroviral packaging and infection, will my infected cells express
tGFP or tRFP?
Answer:No.TheCMV-tGFPandtheCMV-tRFPelementsareoutsideoftheregion
thatgetspackagedbyretrovirus.Thus,youcannotusetGFPortRFPexpressionto
monitortransductionbutyoucanusepuromycinselectiontogeneratestablecell
lines.
Your HuSH product is stated to be locus-specifc. How do I know that it will
knock down the expression of my variant or isoform?
Answer:Unlessstatedotherwise,shRNAconstructsweredesignedtobeeffec-
tiveagainstmosttranscriptionalvariantsataparticulargenelocus.Ifyouwould
likeaknockdownconstructagainstaspecifctranscriptionalvariant(s),OriGene
cangenerateacustomHuSHproductthatwillselectivelyknockdownonlythe
specifedvariants.PleasereviewtheoptionsonourExactHuSHwebsiteatwww.
origene.com/rnai/exact-HuSH.aspx
Can I screen all the constructs provided in HEK293 cells and then pick the
most effective one for subsequent studies?
Answer:Absolutely.Werecommendthatyouscreenallconstructsindividuallyto
identifythemosteffectiveconstructs.HEK293cellsareaconvenientandeasily
transfectablesystemforscreeningallhumanshRNAconstructs(useNIH3T3for
mouseshRNAconstructsorOLN-93forratshRNAconstructs).Othercelllinescan
alsobeusedforthispurpose,solongasthetransfectioneffciencyofourpRS.
pGFP-V-RSandpRFP-C-RSvectorishigh,~70%orgreaterfortransienttransfec-
I6
tions.Afterwards,theeffectiveconstruct(s)canbeusedforretroviralinfectionof
orfordirecttransfectionintoyourtargetcells.
Can I get a HuSH construct against any species other than those on your web
site?
Answer:OriGenecandoanycustomHuSHdesignandconstruction,regardless
ofspeciesspecifcity.Theonlyinformationyouneedtoprovideistheaccession
numberofsequenceyouwishtotarget.Wecanofferyouassistanceinidentifying
theparticularspeciessequencestobeusedforgeneknockdownstudiesoryou
canfollowthelinktoourExactHuSHpagehttp://www.origene.com/rnai/exact-
HuSH.aspx
Will your HuSH products work with any retroviral packaging cell line?
Answer:OurpRS,pGFP-V-RSandpRFP-C-RSvectorshavebeendesignedforviral
packaginginmostcommerciallyavailablepackagingcelllines.However,please
makesurethatthepackaginglinehasnotbeenpreviouslytransfectedwithplas-
midscontainingapuromycinresistancecassette.Furthermore,youneedtoen-
surethatthechosenpackaginglinesviralparticlesareabletoinfectyourtarget
cellline(somecelllineshaverestrictedspeciesspecifcity).Wesuggestpackaging
linessuchasPT67(Clontech)orPhoenix(Orbigen)forusewithourconstructsfor
celllineinfection.
How should I use the products?
Answer:Customerscanusethe5ugshRNADNAplasmiddirectlyfortransfec-
tionandgene-knockdownstudies.Aftertransfection,celllysatescanbeobtained
andusedforWesternblotanalysiswithanantibodyagainstthetargetprotein
toverifythefunctionalityoftheshRNAvectors,orRNAcanbeharvestedfrom
transfectedcellsandusedinquantitativeRT-PCRtodeterminethelossofgene
expression.Ifdesired,theshRNAplasmidscanbere-transformedforunlimited
suppliesoftheplasmids.
Can I select for the plasmid-transfected cells?
Answer:Yes.Onedayaftertransfection,thecellscanbeselectedwithculture
mediacontaining0.5-1.0ug/mLpuromycin(Sigma).
How can I create a stable cell line with a functional shRNA expression vec-
tor?
Answer:Stablecelllinescanbegeneratedbytwodifferentmethods.First,target
cellscanbetransfectedwiththeshRNAplasmid.Onedayaftertransfection,the
transfectedcellscanbeselectedwith0.5-1.0ug/mlpuromycinfor1-2weekswith
passagesasneeded.Alternatively,retroviralpackagingcelllinescanbeusedto
generateretrovirusesforstablecelllinegeneration.Forexample,theplasmidcan
betransientlytransfectedintothePhoenixhelper-freeamphotropicpackaging
cellsline.Twodaysafterthetransfection,thecellculturesupernatantiscollected,
flteredandusedtoinfectthetargetcelllines.Stablecelllinescanbeestablished
followingdrugselection.
II
What use does the empty vector serve?
Answer:ForanyexperimentinvolvingtheintroductionofforeignDNAintocells,
itisimportanttoeliminateanynon-specifceffectsbyusinganemptyvec-
tornegativecontrol.ThevectorscanalsobeusedforcloningyourownshRNA
construct(s).
What use does the scrambled non-effective plasmid serve?
Answer:Tospecifcallyruleoutthepotentialnon-specifceffectinducedbyexpres-
sionoftheHuSHproduct,OriGeneprovidescustomerswithanegativecontrol
(TR30012,TR30013orTR30015),thatwasconstructedbycloningascrambled
sequencecassette(5GCACTACCAGAGCTAACTCAGATAGTACT3)intoourpRS,
pGFP-V-RS,orpRFP-C-RSvectorsrespectively.Theplasmidshouldserveasanega-
tivecontrolforgene-specifcknockdownexperimentsandexcludeanypotential
interferonresponse.
Can you tell me the sequence of your control constructs?
Answer:The29mersequenceusedtotargetfrefyluciferaseinTR30002is5
GGATTTCAGTCGATGTACACGTTCGTCAC3.The29mersequenceusedtotarget
eGFPinTR30001is5CACAAGCTGGAGTACAACTACAACAGCCA3,the29mer
sequenceusedtotargettGFPinTR30009andTR30016is5`GCTACGGCTTCTAC-
CACTTCGGCACCTAC3`,andthe29mersequenceusedtotargettRFPinTR30017
is5CTTCAAGACCACATACAGATCCAAGAAAC3`.Thenon-effectivecontrolse-
quenceis5GCACTACCAGAGCTAACTCAGATAGTACT3.
Do I need to use a special strain of E.coli to amplify my HuSH constructs?
Answer:SpecialE.coliarenotrequiredforHuSHamplifcationbutwedonotrec-
ommendusingJM109.WeroutinelyuseDH5alphafromNewEnglandBiolabs.
Are the HuSH plasmids high copy or low copy number?
Answer:Althoughtheplasmidstechnicallycontainahigh-copynumberOriof
replication,thehairpinslowsreplicationandthus,werecommendusingalow-copy
numbermethodforplasmidDNAamplifcation.
Can the pRS, pGFP-V-RS or pRFP-C-RS vectors be packaged by lentivirus?
Answer:No,ourcurrentRSsystemisstrictlyretroviral.However,weareinthepro-
cessofcreatingalentiviralHuSHvector.Pleasecheckourwebsiteforitsrelease.
Do I have to use retroviral packaging and infection to create stable cell lines?
Answer:Ifyourtransfectioneffciencyisveryhigh(e.g.80%orgreater),itisnot
necessarytouseretroviralpackaging.Simplysplityourcells24hrs.post-transfec-
tionandaddpuromycin(0.5-1ug/ml)tothefreshgrowthmedium.
Will 0.5ug/ml-1ug/ml concentration of puromycin work for my cell line?
Answer:Westronglyrecommendthatakill-curvebeperformedoneachbatchof
cellstoensurethattheoptimalpuromycinconcentrationisemployed.
I8
What is the OriGene guarantee on the shRNA expression plasmids?
Answer:OriGeneguaranteesthatthesequencesintheshRNAexpressioncas-
settesareverifedtocorrespondtothetargetgenewith100%identity.Oneofthe
fourconstructsatminimumareguaranteedtoproduce70%ormoregeneexpres-
sionknock-downprovidedaminimumtransfectioneffciencyof80%isachieved.
WesternBlotdataisrecommendedoverqPCRtoevaluatethesilencingeffectofthe
shRNAconstructs72hrsposttransfection.Toproperlyassessknockdown,thegene
expressionlevelfromtheincludedscramblecontrolvectormustbeusedincompari-
sonwiththetarget-specifcshRNAtransfectedsamples.
Fornon-conformingshRNA,requestsforreplacementproductmustbemadewithin
ninety(90)daysfromthedateofdeliveryoftheshRNAkit.Toarrangeforafree
replacementwithnewlydesignedconstructs,pleasecontactTechnicalServicesat
techsupport@origene.com.Pleaseprovideyourdataindicatingthetransfectioneff-
ciencyandmeasurementofgeneexpressionknockdowncomparedtothescrambled
shRNAcontrol(WesternBlotdatapreferred).
What if the gene is not expressed in HEK293 cells and the transfection eff-
ciencyofmytargetcellsisbelow80%?HowdoIscreenmyshRNAconstructsto
pickthemosteffectiveone?
Answer:IfyourgeneisnotexpressedinHEK293,youcandoaco-transfection
withanexpressionconstructandtheshRNAconstructata1:1ratiotransientlyinto
HEK293cells.Youcanalsoselectstablecellsinyourtargetcellline.Itiswellknown
thatifagenethatisvitalforcellgrowthissilenced,itwillbediffculttogetstable
cellsforthatparticularcellline.
Which method do you recommend for assessing the knockdown effciency of my
gene-specifc shRNA constructs?
Answer:WesternBlotisrecommendedoverqPCRtoevaluatethesilencingeffectof
theshRNAconstructs72hrsposttransfection.Toproperlyassessknockdown,the
geneexpressionlevelfromtheincludedscramblecontrolvectormustbeusedin
comparisonwiththetarget-specifcshRNAtransfectedsamples.
referenceS
BrummelkampTR,BernardsR,AgamiR.(2002)Asystemforstableexpressionof
shortinterferingRNAsinmammaliancells.Science.296:550-3.
ElbashirSM,HarborthJ,LendeckelW,YalcinA,WeberK,TuschlT.(2002)Du-
plexesof21-nucleotideRNAsmediateRNAinterferenceinculturedmammalian
cells.Nature411:494-8.
HannonGJ.(2002)RNAinterference.Nature.418:244-51
LuoB,HeardAD,LodishHF.(2004)SmallinterferingRNAproductionbyenzy-
maticengineeringofDNA.ProcNatlAcadSciUSA.101:5494-9.
PaddisonPJ,CaudyAA,BernsteinE,HannonGJ,ConklinDS.(2002)Shorthairpin
RNAs(shRNAs)inducesequence-specifcsilencinginmammaliancells.GenesDev.
1.
2.
3.
4.
5.
I9
16:948-58.
SenG,WehrmanTS,MyersJW,BlauHM.(2004)Restrictionenzyme-generated
siRNA(REGS)vectorsandlibraries.NatGenet.36:183-9.
ShiraneD,SugaoK,NamikiS,TanabeM,IinoM,HiroseK.(2004)Enzymaticpro-
ductionofRNAilibrariesfromcDNAs.NatGenet.36:190-6.
6.
7.