3Gene Expression Analysis Technologies >10,000Real-time PCR100Low-densityArraysNumber of samples10High-densityArraysRPANorthernSAGE12-10>10,000Number of genesTo consider: Ratio samples / genes, needs for accuracy
4Comparison of Quantitative Assays (RNA/DNA): SensitivityDynamic RangeReal-Time PCRAmplicor/TMA NASBAbDNAXPLOREMicroarraysRPANorthern100101102103104105106107108108107106105104103102101100NASBA: nucleic acid seq based amplificationbDNA: branched DNA assayXplore: based on Invader technologyTMA: transcription mediated amplificationRPA: RNAse protection assayAdvantage: Real-Time PCR
6Phases of amplification Exponential phasePlateau phase
7Exponential phase vs. plateau At some time or another, all reactions regardless of initial amount reach the same plateau!Plateau is not quantitativeExponential phase is quantitativeCyclesAmpliconamountPlateau information is not quantitative
8Before the Real-Time era: End-point PCR Examples of semi-quantitative PCREnd point analysis after reduce number of cycles (exponential phase)Not accurateNot sensitiveCompetitive PCRTime and reagent consumingGOI (pg)0.1110?PCR condition95oC for 2 min# of cycles oC for 15 sec62oC for 30 sec72oC for 30 secEnd Point is at best semi-quantitative
11One Step or Two Step RT-PCR? One Step RT-PCR(One tube)Two Step RT-PCR(Two tubes)RNA1 targetX targetsOligo dTRandom Primers(GS Primers)GS primerscDNATarget pool1 ampliconAmpliconAmpliconX amplicons
12Two-step RT-PCR is more convenient and cost effective One Step or Two Step RT-PCR?One-Step RT-PCRHighly defined conditions to support RT and TaqRequires gene specific primerIdeal for quantification of 1 or 2 messages from a large number of RNA samplesTwo-Step RT-PCRSeparate conditions for cDNA synthesis & PCRFlexible choice of primersIdeal for quantification of multiple genes from a limited number of RNA samplesPerfect for:- Lot of samplesSmall amount of targetsPerfect for:- Few samples- Large amount of targetsTwo-step RT-PCR is more convenient and cost effective
14Good Reverse transcriptase is essential! What RT enzyme?No RNA template degradation > Higher cDNA yieldImproved sensitivityReduced 5’ / 3’ bias due to misprimingIncreased reliabilityGreater percentage of full-length cDNAcomplete sequence representationImproved thermostabilityhigh temperature cDNA synthesisrelaxes secondary structureimproved primer specificityConsistent cDNA synthesis efficiencywide template diversitywide range of template amountRNAse H reduced Thermostable ReverseTranscriptase: SuperScript III ™ RTGood Reverse transcriptase is essential!
17Platinum® Taq DNA Polymerase Which Taq polymerase?Hot Start- Increased specificity- Reduced artifacts (less Primer Dimers)Antibody Hot Start- Fast activation- Maximum activity in early cyclesPlatinum® Taq DNA PolymeraseHot Start Taq is a must!
18PLATINUM® Taq automatic hot start Initial TemplateDenaturationPCR AssemblyTemperature Cycling94oC, 30s - 3 minInactiveTaq DNA PolymeraseFully ActiveTaq DNA Polymerase
22Detection chemistry? Decision, Decision… dsDNA specific stains Ethidium bromide (used in first experiments)SYBR Green I (most widely used today)Probe-Based Systems (unlabeled primers plus probes)TaqMan and TaqMan MGB probe system (5’ nuclease assay)Dual Probe System, uses FRET between probes hybridized side by side to the ampliconMolecular Beacon probe (hairpin probe, quencher and fluorophore are separated when hybridized to the amplicon)Epoch probe (second structure probe, quencher and fluorophore are separated when hybridized to the amplicon)Primer/oligo labeled systemsAmplifluor: hairpin primer with fluorophore and quencherLUX™: primer with one fluorophore and no quencher, proprietary to InvitrogenDecision, Decision…
23Detection chemistry? SYBR Green I® TaqMan® Amplifluor™ LUX™ Quantiprobe®Amplifluor™QLUX™
24Which detection system? MonoplexingCost savingFast initial screeningSybr Green I®- Multiplexing- High Specificity- High SensitivityProbe basedorLUX™ primersA detection system for every applications!
25Light Upon eXtension! LUXTM (Light Upon eXtension) LUX™ The fluorescent intensity is modulated due to the proximity of the fluorophore to specific primary & secondary structures of the oligonucleotide. The design rules have been incorporated into the LUX Designer™. The labeled primer exhibits low fluorescence but incorporation during PCR produces a large increase in fluorescenceLight Upon eXtension!
26Quenching effectThe fluorogenic LUX primer has an attached fluorophore at the 3’end and a short sequence tail on the 5’ end that is complementary to the 3’end of the primer. The resulting hairpin secondary structure provides optimal quenching of the attached fluorophore.The quenching of fluorescence in duplex (ds DNA) is provided by the terminal dG-dC or dC-dG base pair when the dye is attached to a thymidine / cytosine within three nucleotides from the 3΄-end.
27Competitive Audit Data Certified LUX primers were compared to TaqMan® Gene Expression Assays from ABI and QuantiTect Gene Expression Assays from QIAGEN using Platinum Super-Mix UDG (1X ROX).Standard curve generated from ten-fold serial dilutions of ORF clone (107 –102 copies).ABI 7700PCR Efficiency: 93%R2:0.998PCR Efficiency: 92%R2:0.999Quantitect IL6PCR Efficiency: 94%R2:0.996LUX IL6TaqMan IL6
28Comparison of LUX™ to TaqMan LUX™ sensitivity advantages
29LUX™ Built-in control feature! Melting curve analysisMethod:Real-time PCRAfter amplification: 60°C to 95°C (slow ramp)Fluorescence signal is recorded continuously during the slow temperature rampMelting curve : Fluorescence signal vs. TemperatureMelting curve is converted to melting peaks by plotting –dF/dT vs TemperatureLUX™ Built-in control feature!
30Advantages LUX™ versus TaqMan® Specificity – Melting CurveFast control of the PCR product specificity without:opening the tube andrunning a gelProvides accurate real time assessment of quality:- eliminates risk of false positivesreduces risk of contamination
31Advantages LUX™ versus TaqMan® Alexa 546TETFAMAll detectorsExcitation(nm)EmissionFAM492517JOE520548TET521536HEX533550Alexa Fluor 546554570Data from Molecular Probes spectraTriplex amplification using standard protocol:- 10,000 copies of Gene X are amplified using a LUX primer set labeled with Alexa Fluor 546- 1,000 copies of Gene Z are amplified using a LUX primer set labeled with TET- 100 copies of Gene Y are amplified using a LUX primer set labeled with FAMEasy multiplexing!
32Advantages LUX™ versus TaqMan® Simple Primer Design with the new D-LUX™ Designer
35From cells to cDNA in ONE TUBE! Cells Direct kitCell LysisNo RNA isolation step required.DNase treatment eliminates genomic DNA so you can be confident that results are due to cDNA amplification.DNase TreatmentAll done in 1 tube!The cDNA synthesis is performed by SuperScript™ III RNase H- RT.cDNA SynthesisApplication:qPCRFrom cells to cDNA in ONE TUBE!
36From 1 cell to 10,000 cells without NAP! Cells Direct kitFrom 1 cell to 10,000 cells without NAP!
37Room Temperature Stable RTS kitsOptimized PCR SuperMixVialingProprietary Polymer MixTemperature-controlled LyophilizationPackagingShelf-life = 1 yearComplete Cycle within 3 daysRoom Temperature Stable
38RTS and regular mixes have equal performances RTS kitsStandard Curve RTS one stepy = (x)R2= 0.998101520253035401.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+06Starting ConcentrationCycle ThresholdStandard Curve aqueous (SuperScript III one step)y = (x)R2= 0.999101520253035401.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+06Starting ConcentrationCycle ThresholdRTS and regular mixes have equal performances
40Invitrogen qPCR Reagent Selection Guide SYBR Green DetectionFluorescent Probes/Primers(LUX™, TaqMan®, etc.)ABI 7000, 7700, 7900Roche LightCyclerAny InstrumentDNA or cDNAPlatinum SYBR Green qPCR SuperMix-UDG w/ROXPlatinum SYBR Green qPCR SuperMix-UDG w/BSAPlatinum SYBR Green qPCR SuperMix-UDGPlatinum Quantitative PCR SuperMix-UDG w/ROXPlatinum Quantitative PCR SuperMix-UDGRNA, 1-StepSuperScript III Platinum SYBR Green One-Step qRT-PCR Kit w/ROXSuperScript III Platinum SYBR Green One-Step qRT-PCR Kit w/BSASuperScript III Platinum SYBR Green One-Step qRT-PCR KitSuperScript III Platinum One-Step qRT-PCR Kit w/ROXSuperScript III Platinum One-Step qRT-PCR KitRNA, 2-StepSuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit w/ROXSuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit w/BSASuperScript III Platinum SYBR Green Two-Step qRT-PCR KitSuperScript III Platinum Two-Step qRT-PCR Kit w/ROXSuperScript III Platinum Two-Step qRT-PCR KitWe will test AM’s on the most suitable kit for the two customer examples outlined earlier.A solution for each problem!
41SYBR Green LC qPCR versus Roche qPCR Title of slide is set as Arial, Bold, Italic, size 24Takeaway Box is also Arial, bold, italic, size 24– type the information first and then draw a gray 2 1/4 pt box around the copy block.All text in ArialOnly italicize Titles and Text Boxes – use Title CaseDon’t do all caps for titles or column headings …use Title CaseNo gray fill in objects where you have black type ... doesn’t copy wellNo shadowing … on anythingKeep graphics very simple … 2D vs. 3D … keep colors compatible throughout presentationKeep text within the boundaries so can be viewed well when on a large screenHPRT primers were used with plasmid standards from 1x107 to 1x101 copies. Invitrogen SYBR Green LC (blue) quantified all 7 logs with a slope of –3.644 and an R-value of No template controls (NTC’s) were negative with the SYBR LC kit.Roche’s FastStart DNA Master Plus (green) quantified 5 of 7 logs with a slope of –3.700 and an R-value of The NTCs along with the last two dilutions showed contamination.
49Invitrogen multidisplinary qPCR group R&D qPCR group in Carlsbad, CAEnzymologists in Carlsbad, CAChemists from Molecular Probes in Eugene, ORCustom Primer manufacturing facility in Frederick, MAEvoQuest service group in Carlsbad, CAAnd more…