Roche X-tremeGENE SiRNA Transfection Reagent Manual.pdf

www.roche-applied-For life science research only.Not for use in diagnostic procedures.X-tremeGENE siRNA Transfection ReagentFor the transfection of siRNA into animal cellsCat.No.04 476 093 001 1 mlCat.No.04 476 115 001 Multi-pack 5?1 ml(2000 transfection)Version June 2006Store at?2 to?8C1.What this Product DoesNumber of Transfection ExperimentsUsing standard experimental conditions,one milliliter of X-tremeGENEsiRNA Transfection Reagent transfects HeLa,NIH 3T3,Hek 293 cells,or other mammalian cells in over four hundred wells of a 24-well plate.This is equivalent to over 1,600 transfections in 96-well plates.ContentsFormulation X-tremeGENE siRNA Transfection Reagent is a propri-etary blend of lipids and other components supplied in aqueous solu-tion,sterile-filtered,and packaged in polypropylene tubes.The reagentis free of any components derived from animals.Storage and StabilityX-tremeGENE siRNA Transfection Reagent is shipped at room tem-perature and is stabilized for extended storage at+2 to+8C throughthe expiration date printed on the label when tightly closed.NAlways mix X-tremeGENE siRNA Transfection Reagent prior to use(vortex for one second or invert).Do not freeze!Additional Equipment and Reagents RequiredRefer to the list below for additional reagents and equipment requiredto transfection experiments.Prewarmed sterile,serum-free culture medium without additives orsupplements.Opti-MEM1 Medium is recommended for dilution ofthe siRNA and the X-tremeGENE siRNA Transfection reagent.Mammalian cell line of interest siRNA solution between 0.2?g/?l(15 pmoles/?l,i.e.15?M)and 2?g/?l(150 pmoles/?l,i.e.150?M)in sterile buffer.ApplicationX-tremeGENE siRNA Transfection Reagent is a multicomponentreagent that forms a complex with siRNA,and then delivers it into ani-mal cells.Features of X-tremeGENE siRNA Transfection Reagent include:High transfection efficiency in many common cell types,includingHeLa,NIH 3T3,Hek 293,PC-3 and COS-7.The use of one single reagent for siRNA-and cotransfection-basedRNA experiments.Low cytotoxicity,the change of media after the addition of transfec-tion complex is not required.Functions exceptionally well in the presence or absence of serum;eliminates the need to change media.2.How To Use this Product2.1Before You BeginsiRNA purity,fluorescently labeled siRNAsDetermine the siRNA purity using a 260 nm/280 nm ratio;the ratioshould be 1.9 or higher.When using siRNA preparations from in vitrotranscription reactions,the size of less than 30 basepairs of the siRNAneeds to be carefully controlled,as non-embryonic animal cells showan interferon response resulting in a general translation block withlarger dsRNA species(1).A recent publication(1)describes that the5tri-phosphate residues of in vitro transcribed siRNA must beremoved to avoid an interferon response.Chemically synthesized si-RNAs from commercial suppliers are usually sufficiently pure fortransfection.NUse fluorescently labeled siRNAs:The uptake or non uptake of flu-orescently labeled siRNAs does not necessarily correlate with theknockdown.Even with no visible uptake of labeled siRNAs,suc-cessful knockdown was demonstrated.Cell-Culture ConditionsMinimize both intra-and inter-experimental variance in transfectionefficiency by using cells that are regularly passaged,proliferating well(best when in a loggrowth phase),and plated at a consistent density.Effect of Media and Media Additives,including Sera?In some cell types,antimicrobial agents(e.g.,antibiotics and fungi-cides)that are commonly included in cell culture media mayadversely affect the transfection efficiency of X-tremeGENE siRNATransfection Reagent.If possible,exclude additives for initial experi-ments.Once high-efficiency conditions have been established,these components can be added back while monitoring your trans-fection results.?Different media and media components may influence the level oftransfection efficiency and subsequent growth of the transfectedcells,as well as knockdown of the gene of interest.LAny medium can be used for cell cultivation and during trans-fection.However,Opti-MEM1 Medium is recommended fordilution of the siRNA and the X-tremeGENE siRNA Transfectionreagent(see Procedure).?Test different media and optimize the level of each medium compo-nent for these effects.Although it is not usually necessary to removethe transfection reagent:siRNA complex following the transfectionstep,it is necessary to feed your cells with fresh media for extendedgrowth periods.This is particularly important when the transfectedcells are allowed to grow for 37 days for maximal knockdown withvery stable proteins.Verification of Transfection EfficiencyAs the knockdown efficiency is dependent on the gene as well as onthe cell line,determination of the transfection efficiency is recom-mended when using a new cell line.The knockdown of the endoge-nous human HPRT housekeeping gene can be measured with Q-PCR.The knockdown of the endogenous lamin A/C gene with anti-laminA/C antibodies(BD Biosciences)was demonstrated in western blots.Incubation Time Incubate the cells for 2472 hours.The length of incubation dependsupon the siRNA,the cell type being transfected,the stability of themRNA,or the protein being targeted.After this incubation period,measure the knockdown using an assay that is appropriate for yoursystem.0606.044922420012www.roche-applied-2.2ProcedurePreparation of Cells for TransfectionOne day prior to the transfection experiment,trypsinize,adjust the cellconcentration,and plate the cells in the chosen cell culture vessel.Formost cell types,plating 0.1 0.8 x 105 cells in a 24-well plate in 0.45 mlof medium overnight will achieve the desired density of 3050%con-fluency.If using culture plates of a different size,adjust the startingvolume of X-tremeGENE siRNA Transfection Reagent and the startingmass of siRNA in proportion to the relative surface area(see table 1).Ratio OverviewPreparation of a complex that is sufficient for a single well of a 24-wellplate at three different concentrations.Preparation of X-tremeGENE siRNA Transfection Reagent:siRNA Complex and Transfection of Cells in a 24-well plateFor initial optimization,use 10:2,2.5:0.5,and 1:0.2 ratios of X-trem-eGENE siRNA Transfection Reagent(l)to siRNA(g),respectively.The preparation of the complex for a single well of a 24-well plate isdescribed below.These ratios will function very well for commonlyused adherent cells.NThe X-tremeGENE siRNA Transfection Reagent:siRNA complexmust be prepared in medium that does not contain serum,even ifthe cells are transfected in the presence of serum.LFor additional optimization tips,go to www.roche-applied-sci- X-tremeGENE siRNA Transfection Reagent with serumfree Opti-MEM-1 medium(without antibiotics or fungicides):NSerum-free medium must be pipetted first.The order andmanner of addition is critical.Label three small sterile tubes:“10,”“2.5,”and“1.”Pipet 40?l of serum-free medium into the first,47.5?l into the second,and 49 l into the last tube.Pipet the X-tremeGENE siRNA Transfection Reagent directly into the medium without allowing contact with the walls of the plastic tube:10?l X-treme GENE siRNA Transfection Reagent into the first tube,and 2.5?l of the reagent into the tube labeled“2.5”and 1?l of the reagent into the tube labeled“1”.Tube labelSFM(serum-free medium)(?l)X-tremeGENE siRNA Transfection Reagent(?l)1040102.547.52.51491 Mix cautiously by pipetting up and down.NDo not vortex!NTo avoid adversely affecting transfection efficiency,do notallow undiluted X tremeGENE siRNA Transfection Reagentto come into contact with plastic surfaces(such as the wallsof the tube containing the serum-free medium)other thanpipette tips.Diluted siRNA should be combined with trans-fection reagent(step 3)within 5 min!?Dilute siRNA with serum-free Opti-MEM-1 medium(without antibiotics or fungicides):NSerum-free medium must be pipetted first.The order andmanner of addition is critical.Label three small sterile tubes:“50+2,”“50+0.5,”and“50+0.2.”Pipet serum-free medium into all tubes to yield a final volume of 50?l.Tube labelSFM(l)+siRNA(?g)Final volume(?l)50+250+2(i.e.150 pmoles)5050+0.550+0.5(i.e.40 pmoles)5050+0.250+0.2(i.e.15 pmoles)50 Pipet the siRNA directly into the medium without allowing con-tact with the walls of the plastic tube:2?g siRNA into the first tube,0.5?g of the siRNA into the tube labeled“50+0.5”and 0.2?g of the siRNA into the tube labeled“50+0.2”.Mix cautiously by pipetting up and down.NDo not vortex!NTo avoid adversely affecting transfection efficiency,do notallow undiluted siRNA to come into contact with plastic sur-faces(such as the walls of the tube containing the serum-free medium)other than pipette tips.Diluted siRNA and should be combined with transfection reagent(step 3)within 5 min!?Mix and incubate the complex:Mix the contents of the tube from Step 1 with that of Step 2 in the following order:-Tube labeled 10“with 50+2“-Tube labeled 2.5“with 50+0.5“-Tube labeled 1“with 50+0.2“Mix cautiously by pipetting up and down.NDo not vortex!Incubate the transfection reagent siRNA complex for 15-20 min at+15 to+25C.Add the entire volume to each well of a 24 well plate.?Add complex to the cells:Remove culture vessel from the incubator.Removal of growth medium is not necessary.Add the transfection reagent:siRNA complex dropwise to the cells,and swirl the wells or flasks cautiously to ensure distribu-tion over the entire plate surface.?Return the cells to the incubator until the assay for gene knock-down is performed.Once the X-tremeGENE siRNA Transfection Reagent:siRNA complex has been added to the cells,there is no need to remove and replace with fresh medium(as is neces-sary with some other transfection reagents).LThe exposure of most common laboratory cell types(COS-7,NIH 3T3,HEK-293,HeLa)to the reagent:siRNA complex untilmeasurement of the gene-knockdown(2472 hours later)does not affect the results.When using serum-free mediumduring the transfection procedure(step 4),replace themedium with serum-containing medium 38 hours aftertransfection.3www.roche-applied-Optimizing Ratio of X-tremeGENE siRNA Transfection Reagent,siRNA and Plasmid DNA in the given Plate FormatesRefer to the table below(table 1)when setting up your transfectionreactions in other plate formates.The starting volume and mass isbased on a X-tremeGENE siRNA Transfection Reagent:siRNA ratio of5:1.The ranges cover very different ratios.When varying the siRNAconcentration,make sure to keep the X-tremeGENE siRNA Transfec-tion Reagent in a ratio of 2-10:1 to the siRNA mass.Table 1 Co-Transfection ExperimentsThe X-tremeGENE siRNA Ttransfection Reagent has the potential forco-transfection of short inhibitory RNAs and plasmid DNA.Usuallytransfecting plasmids requires a higher cell density at the point oftransfection compared to siRNA.Follow the suggestions when per-forming co-transfection experiments:Plate cells such that they reach 90%confluency at the time of trans-fection Maintain the same total reagent:total nucleic acid ratio as thatused for siRNA alone in your system.(If you need to increase thetotal amount of nucleic acid,then increase the amount of transfec-tion reagent in proportion to the total amount?g of nucleic acid)Use the table below(table 2)for optimizing X-tremeGENE siRNATransfection Reagent,siRNA and plasmid DNA in the given plateformatesNAlways use a volume of X-tremeGENE siRNA TransfectionReagent that is at least 3-fold in excess of the total final mass ofnucleic acid.Table 2Culture plate diametermm96-well plate24-well plate6-well plateSurface area cm20.41.99Plated cells?105suggested for start0.05-0.20.10.1-0.80.41-42Medium volume ml0.150.452.0siRNA?g rangesuggested for start?gDilution volume?l0.05-0.30.15in 150.1-1.00.5(40 pmoles)in 500.4-5.02.0in 100X-tremeGENE siRNA Trans-fection Reagent?lsuggested for start?lDilution volume?l0.1-4.00.8in 150.5-102.5in 502-3510in 100Total volume ml 0.180.552.2Culture plate diameter mm96-well plate24-well plate 6-well plateSurface area cm20.41.99Plated cells?105suggested for start0.05-0.20.10.1-0.80.41-42Medium volume ml0.150.452.0siRNA?g rangesuggested for start?g0.05-0.30.080.1-1.00.250.4-5.01.0DNA?g0.150.52.0Nucleic acid dilution volume1550100X-tremeGENE siRNA Trans-fection Reagent?lsuggested for start?lDilution volume?l0.1-4.00.8in 150.5-102.5in 502-3510in 100Total volume ml0.180.552.23.TroubleshootingObservationsPossible CauseRecommendationLow/No knockdown levels observedLow transfection efficiencyCells were grown con-fluent at the time of transfection.Seed cells at a lower density that they reach 30-50%confluency at the time of transfection.Cells passaged too often or used from sta-tionary phase.Use cells with low passage number and only cell cultures that were regu-larly passaged at log phase.Not enough siRNA used.Increase the amount of siRNA.Not enough X-treme GENE siRNA Transfec-tion Reagent used.Optimize transfection conditions for each cell line by varying the amount of siRNA:X-treme GENE transfec-tion complex and the ratio of siRNA:X-treme GENE siRNA Transfection Reagent.Antibiotics added dur-ing transfection.Avoid the addition of antibiotics dur-ing transfection.Serum present during siRNA:X-tremeGENE complex formation.Use serum-free medium during com-plex formation.siRNA not activeTarget sequence not suited.Select another target region.d-siRNA degradedCheck integrity of siRNA on poly-acrylamide or agarose gels.Do not store siRNA in water.Use a sterile RNAse free buffer con-taining 10 mM Tris,pH 8.0,20 mM NaCl,1 mM EDTA for storage.Store siRNA aliquoted at-15 to-25C and avoid repeated freeze/thaw cycles.Large cellular amounts or high stability of the targeted mRNA or pro-teinPerform a time course experiment and determine the time when the highest degree of knockdown is obtained.Perform qPCR analysis(e.g.,by LightCycler)to measure mRNA lev-els when only low knock down on protein level isobserved.Repeat the addition of siRNA:X-tremeGENE transfection complex and refresh medium forvery long-lived target protein species.Cytotoxic effects after transfection observedToo much X-treme-GENE siRNA Transfec-tion Reagent used.Reduce/optimize amounts of X-tremeGENE siRNA Transfection Reagent for each cell line.Cells are very sensitive to transfectionRemove transfection medium and add new prewarmed serum contain-ing medium after 4-6 hours.(This will not reduce transfection efficiency.)Unpurified d-siRNA used for transfectionTo avoid a general translation block and initiation of apoptosis,carefully purified siRNA of less than 30 nucle-otides are necessary for most somatic mammalian cell lines(7,8).Non-specific off-target gene knockdown observedTarget sequence siRNA sense or antisense strand contains strong homology to other genes.Select a new target sequence.Lower the concentration of the siRNALimit the size of the target sequence to 1.0 kb when using d-siRNA.Roche Diagnostics GmbHRoche Applied Science68298 MannheimGermany4.Additional Information on this ProductBackground In