myiq and iq 5 - bio-radthermal gradient experiment for optimizing annealing temperature. a 10-fold...
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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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Volume 10µl
Avg.CT 19.84
SD 0.11
Volume 25µl
Avg.CT 20.97
SD 0.11
EmissionfilterwheelCCD
Foldmirror
Excitationfilterwheel
Lens
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.