Amplification: MyiQ and iQ5 Real-Time PCR Systems

MyiQ™and iQ™5 Real-TimePCRDetectionSystems

Genomic Research Solutions From Bio-RadBio-Radiswellknownformakingadvancedgenomictechnologies

accessibletoeveryresearcher.Inadditiontoproducingoutstanding

instrumentationforreal-timePCRdetection,Bio-Radoffersavarietyof

solutionsforsimplifyingeverythingfromassaydesigntogeneexpression

analysis.Thiscompletecollectionoftoolsprovidespowerfulbuilding

blocksforyourgenomicdiscoveries.

Bio-Rad Offers Real-Time Instruments Designed for the Way You’d Rather Workn Modularinstrumentswithhigh-resolutionopticsformaximumflexibilityandsensitivity

n Gradientfunctionalityforrapidassayoptimization

n OutstandingthermalperformancedeliveredbytheiCycler®thermalcycler

n PCRsupermixesthatprovideoptimumperformanceinreal-timePCRassays

n Preciselymanufacturedplates,tubes,andsealerstestedforoptimalfitandperformanceinBio-Radreal-timePCRinstruments

Products for Your Genomics Workflow

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BeaconDesignersoftwarefacilitatesthedesignandselectionofprimersandprobesforavarietyofreal-timePCRapplications.

Aurum™RNAisolationkitsyieldfastresultswiththehighestlevelofsamplepurity.

TheExperion™automatedelectrophoresissystemquicklyandaccuratelymeasuresRNAquality.

AbroadvarietyofPCRreagentsandinstrumentsprovidetheflexibilityandreliabilityyouneedtoacceleratediscovery.

Assay

Design

Sample

Preparation

Sample Quality

Assessment

Real-Time

Amplification

and Analysis

Smart Modular Designs TheMyiQandiQ5real-timePCRdetectionsystemsare96-wellmodularupgradestotheiCyclerthermalcycler,whichdeliversexcellentthermalperformanceandthermalgradientcapability.Sensitivecharge-coupleddevice(CCD)-basedopticsenableaccuratequantitationoveradynamicrangeof9ordersofmagnitude.

MyiQ SystemTheMyiQsystemoffersanaffordableoptionforthedetectionofcommongreenfluorescentdyes,suchasFAMandSYBRGreenI.

iQ5 SystemTheiQ5systemoffersfive-targetanalysiscapabilitiesformultiplexPCRwithavarietyofdetectionchemistries.

ThecombinedperformanceoftheiCyclerthermalcycler,theCCD-basedoptics,andBio-Radsupermixesgeneratesexceptionallyuniformresultsacrossa96-wellblock.Withreactionvolumesaslowas10µl,theiQ5andMyiQsystemsproduceuniformdatawithstandarddeviationsof<0.15cycles(seedatabelow).

Optical system.Excitationofall96wellsisachievedbyacombinationofnarrow-bandpassfiltersandatungsten-halogenlamp.Filteredlightfromthelampisreflectedoffmirrors,passesthroughacondensinglens,andisfocusedintothecenterofeachwell.Fluorescentlightemittedfromthewellsreflectsoffthemainfoldmirror,passesthroughanemissionfilter,andisdetectedbya12-bitCCD.

Excellent uniformity.IL1-bplasmidtemplatewasdilutedto105copies/reactionandamplifiedinthepresenceofaFAM-labeleddetectionprobewithiQ™supermixusinganiQ5real-timePCRdetectionsystem.Leftpanel,replicatesof10µlreactions.Rightpanel,replicatesof25µlreactions.Inset:CT,thresholdcycle;SD,standarddeviation.

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Avg.CT 19.84

SD 0.11

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Avg.CT 20.97

SD 0.11

EmissionfilterwheelCCD

Foldmirror

Excitationfilterwheel

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96-wellplate

Optimize Reactions in a Single ExperimentBio-Radreal-timesystemsofferathermalgradientfeature,whichallowsyoutooptimizeassaysinasingleexperimentbyusingarangeoftemperaturessimultaneously.Atanystepinaprotocol,atemperaturegradientofupto25°Cmaybeprogrammedacrossthereactionblock.

n Gradientcontrolprovidesexceptional uniformityandreproducibilitywithineachzone

n Easyprogrammingwithonscreenpresentationofgradienttemperatures

n Precisionramping

n Availableforbothconventionaland quantitativePCRinstrumentation

n Eachtemperaturewithinthethermalgradientislistedinvalidationreports

Outstanding Gradient Performance

Thermal gradient.Optimizingincubationtemperaturesforreal-timePCRassaysiscritical,particularlyformultiplexassays,butitisnotalwayseasytodo.Afterthemeltingtemperature(Tm)ofaprimeriscalculated,theannealingtemperaturemustbedeterminedempiricallyforoptimalresults.Thisofteninvolvesrepeatingareactionatmanydifferenttemperatures.Similartime-consumingtestsmayberequiredtooptimizethedenaturationtemperature.Thethermalgradientfeaturefacilitatesidentificationofthemostfavorabletemperaturesforoptimalassayperformance.

Thermal gradient experiment for optimizing annealing temperature.A10-folddilutionseries(108–102copies)ofplasmidcontainingGAPDHtemplatewasamplifiedusingathermalgradientinthepresenceofSYBRGreenIdye.TheamplificationreactionwasmonitoredandanalyzedwithaniQ5real-timePCRdetectionsystem.Eightidenticalreactionswerepreparedforeachtemplateconcentration,oneforeachoftheeightPCRannealingtemperatures,rangingfrom55to70°C.Anoptimalprimerannealingtemperatureof58°C(showninthegreentraces),whichresultedintheearliestCTvalue,wasidentifiedinthisgradientassay.Thebluetracesshowtheresultsfor64.5°Casacomparison.

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Makingthemostofapowerfulreal-timePCRsystemrequiresaflexibleandeasy-to-usesoftwarepackage.TheiQ5opticalsystemsoftwaremeetsthisneedwithquick-setuptools,afullsuiteofanalysistools,andavarietyofpresentationoptions.Inaddition,iQ5opticalsystemsoftware,SecurityEdition,providesthetoolsrequiredtomeetregulatoryrequirements.

StandardfeaturesofiQ5softwareinclude:

n Advancedgeneexpressionanalysisoptions,includingcomparisontomultiplereferencegenes,correctionforreactionefficiency,andmore

n AbilitytoexportanydatacollectedandanalyzedbyiQ5softwaredirectlytoaMicrosoftExcelspreadsheet

n GeneStudymoduleformultifilegeneexpressionanalysistodirectlycompareover5,000CTvaluesfromiQ5andMyiQdata

n UserProfilemanagementtoolforcreatinguniqueuserlog-innames,withoptionalpasswordprotection,forfilestorageanddataanalysispreferences

n Built-inanalysismodulesforabsolutequantitation,melt-curveanalysis,end-pointanalysis,andallelicdiscrimination(formultiplexdata)

Security for Those Who Need ItTheSecurityEditionofiQ5opticalsystemsoftwarecanintegratethepowerofaniQ5real-timePCRdetectionsystemwithyourlaboratory’sgoodlaboratorypractice(GLP)standardsfordatacollectionanddataanalysis.Thissoftwareisdesignedforroutineusebyscientistswhooperateinregulatedenvironmentsthatrequireelectronicrecord-keepingprocedures.

TheSecurityEditionoffersseveralmajorfunctionsandfeatures,including:

n EmbeddedtoolsforcompliancewithUSFDA21CFRPart11regulations,includingelectronicsignatureandaddedtraceabilityfeatures

n Enhancedreportingtoolsforcustomizingthesecurepresentationofassayresults

Multifile gene expression analysis.CompareCTvaluesfromdifferentdatafileswiththeGeneStudyiniQ5software.Over5,000CTvaluescanbeanalyzedinasingleGeneStudyfile.

Efficient, Accurate, Easy-to-Use Software

Electronic record-keeping tools.TheiQ5SecurityEditionsoftwareenableselectronicsignaturestobeappliedtocalibrationanddatafilesgeneratedbyiQ5orMyiQsystems.

Bio-Radreagentsaccentuatetheperformanceofyourreal-timesystem.AllreagentsdemonstratehighperformancewithminimaloptimizationoverawidedynamicrangeforinputRNA,cDNA,genomicDNA,andplasmidDNA.

Detect Multiple Targets Without OptimizationiQmultiplexpowermixisarobustmixthatgreatlysimplifiesthechallengeofidentifyingreactionconditionsthatwillamplifytargetswithequalefficiencyinsingleplexandmultiplexreal-timePCRreactions.Thismixmakesmultiplexreal-timePCReasierbyremovingtheneedtooptimizebuffer,enzyme,orprimerconcentrations.

Convenient One-Step RT-qPCR for Any Detection ChemistryiScript™one-stepRT-PCRkitsareoptimizedtodelivermaximumRT-PCRefficiency,sensitivity,andspecificity.ThesekitscontainaproprietaryreactionbufferthathasbeenspecificallyformulatedtooptimizeactivityofbothiScriptreversetranscriptaseandiTaq™DNApolymerase,whileminimizingthepotentialforprimer-dimerformationandothernonspecificPCRartifacts.Withthesekits,cleandetectionoflow-copytargetsiseasytoachieve.

Protect Precious Samples With High-Quality Plastic ConsumablesBio-Radoffershigh-qualityconsumablesforawidevarietyofapplications,backedbytechnicalsupportprofessionals.

EachboxofBio-Radtubes,plates,andcapsisprocess-sampledandtestedtobenegativeforDNase,RNase,andDNA.AllreactionvesselsandsealingsystemshavebeendesignedtoprovidethebestpossiblefitandperformancewithacorrespondingBio-Radthermalcycler.Forreal-timePCRapplications,Bio-Radofferspreciselymanufacturedtubes,plates,sealers,andaccessoriesthathavebeentestedforoptimalperformanceiniQ5andMyiQsystems.

Optimized Reagents for Peak Performance

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An accurate one-cycle spacing between CT values is precisely maintained in a series of 2-fold dilutions. HumangenomicDNA(120–15ng)wasamplifiedwithiQsupermixusingprimersandaprobespecifictotheIL-1bgene.Eightreplicatesateachtemplateconcentrationwereamplifiedalongwithno-templatecontrolsontheMyiQreal-timesystem.Standardcurvehadr=–0.999,slope=–3.378,efficiency=97.7%.

Linearity of five-target multiplex detection with the iQ5 real-time PCR detection system.A–E,fluorescencedatafromaseriesof10-folddilutionsofplasmidDNA(108–102copiesper25µlreaction)amplifiedusingfivereporterdyestomonitorfivetargets,asindicatedabovepanels.F,standardcurvesgeneratedfromdatainA–E:FAM,R2=0.998,efficiency=98.0%;HEX,R2=0.999,efficiency=104.4%;TexasRed,R2=0.999,efficiency=102.5%;Cy5,R2=0.999,efficiency=98.9%;TAMRA,R2=0.999,efficiency=100.8%.

A. FAM (b-actin)

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B. HEX (GAPDH)

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C. Texas Red (IL-1b)

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F. Standard curvesE. TAMRA (cyclophilin)

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iQ™ SYBR® Green supermix.

•Standard6Unknown

308.78

49.56

144.82

127.52103.47

Life ScienceGroup

06-0317 0307 Sig 1106Bulletin 5462 US/EG Rev A

Bio-Rad Laboratories, Inc.

Web site www.bio-rad.com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400 Canada 905 712 2771 China 86 21 6426 0808 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000

System Specifications

TemperatureRange 4–100°CAccuracy ±0.3°CofprogrammedtargetUniformity ±0.4°Cwell-to-well

Gradient ModeGradientaccuracy ±0.4°CRowuniformity ±0.4°CGradientrange 40–100°CTemperaturedifferential 1–25°C range

Optical SystemLightsource Tungsten-halogenlamp

Fluorescenceexcitationrange MyiQ 475–495nm iQ5 475–645nm

Fluorescencedetectionrange MyiQ 515–545nm iQ5 515–700nm

Sensitivity OnecopyofIL-1binhumangenomicDNA

Dynamicrange 9ordersofmagnitude

Compatibledyes MyiQ FAM,SYBRGreenI iQ5 FAM,SYBRGreenI,HEX,TAMRA, TexasRed,Cy5

DescriptiveSamplecapacity 96wellsSamplevolume 15–100µlDimensions(WxDxH) 29.2x58.4x38.7cm(11.5x23x15.2")Weight 17.6kg(39lb)

Ordering InformationCatalog# Description170-9770 MyiQ Single-Color Real-Time PCR Detection

System,includesiCyclerbase,opticsmodule,softwareCD-ROM,96-wellopticalreactionmodule,optical-quality96-wellPCRplates,Microseal®'B'seals,communicationcables,powercords,instructions

170-9780 iQ5 Multicolor Real-Time PCR Detection System,includesiCyclerbase,opticsmodule,softwareCD-ROM,5installedfiltersets,96-wellreactionmodule,calibrationsolutions,optical-quality96-wellPCRplates,Microseal'B'seals,communicationcables,powercord,quickreferencecards,instructions

170-8734 Beacon Designer Probe/Primer Design Software,includesCD-ROM,quickguide,instructions

170-9753SE01 iQ5 Optical System Software, SecurityEdition,single-seatlicense

170-9799 Real-Time PCR Applications Guide223-9441 iQ 96-Well PCR Plates,25

Catalog# DescriptionTBS-0201 0.2 ml 8-Tube Strips Without Caps,natural,125TCS-0803 Optical Flat 8-Cap Strips,for0.2mltubesandplates,

ultraclear,120MSB-1001 Microseal 'B' Adhesive Seals,100170-8848 iQ Multiplex Powermix,50x50µlreactions,2xmix

containsdNTPs(includingdUTP),11mMMgCl2,iTaqDNApolymerase,stabilizers

170-8860 iQ Supermix,100x50µlreactions,2xmixcontains100mMKCl,40mMTris-HCl,pH8.4,0.4mMeachdNTP(dATP,dCTP,dGTP,dTTP),50U/mliTaqDNApolymerase,6mMMgCl2,stabilizers

170-8875 iTaq DNA Polymerase,5U/µl,includes5,000Upolymerase,25mlof10xPCRbuffer,25mlof50mMMgCl2solution

170-8894 iScript One-Step RT-PCR Kit for Probes,50x50µlreactions,includesiScriptreversetranscriptaseforone-stepRT-PCR,2xprobesRT-PCRreactionmix,nuclease-freewater

732-6800 Aurum Total RNA 96 Kit,2x96-wellpreps,includes2growblocks,2growthmembranes,sealingtape,2RNAbindingplates,2collectionmicroplates,2vialslyophilizedDNaseI,RNase-freereagents,protocoloverview,instructions

700-7000 Experion System,100–120/220–240V,forproteinanalysis,includeselectrophoresisstation,primingstation,software,USB2cable,instructions(analysiskitssoldseparately)

BeaconDesignerisatrademarkofPREMIERBiosoftInternational.CyisatrademarkofGEHealthcare.ExcelandMicrosoftaretrademarksofMicrosoftCorp.SYBRandTexasRedaretrademarksofMolecularProbes,Inc.Bio-RadLaboratories,Inc.islicensedbyMolecularProbes,Inc.tosellreagentscontainingSYBRGreenIforuseinreal-timePCR,forresearchpurposesonly.

MolecularbeaconsandtheirusearelicensedunderpatentsownedbyThePublicHealthResearchInstituteoftheCityofNewYork,Inc.

NoticeregardingBio-Radthermalcyclersandreal-timesystems.Purchaseofthisinstrumentconveysalimitednon-transferableimmunityfromsuitforthepurchaser’sowninternalresearchanddevelopmentandforuseinappliedfieldsotherthanHumanInVitroDiagnosticsunderoneormoreofU.S.PatentsNos.5,656,493,5,333,675,5,475,610(claims1,44,158,160–163,and167only),and6,703,236(claims1–7only),orcorrespondingclaimsintheirnon-U.S.counterparts,ownedbyAppleraCorporation.Norightisconveyedexpressly,byimplicationorbyestoppelunderanyotherpatentclaim,suchasclaimstoapparatus,reagents,kits,ormethodssuchas5'nucleasemethods.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.

Bio-Rad’sreal-timethermalcyclersarelicensedreal-timethermalcyclersunderApplera’sUnitedStatesPatentNo.6,814,934B1foruseinresearchandforallotherfieldsexceptthefieldsofhumandiagnosticsandveterinarydiagnostics.

LabChipandtheLabChiplogoaretrademarksofCaliperLifeSciences,Inc.Bio-RadLaboratories,Inc.islicensedbyCaliperLifeSciences,Inc.

tosellproductsusingtheLabChiptechnologyforresearchuseonly.