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Real-Time quantitative PCR: Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005.

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Presentation on theme: "Real-Time quantitative PCR: Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005."— Presentation transcript:

1 Real-Time quantitative PCR: Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005

2 Invitrogen Proprietary and Confidential Why using real-time PCR?

3 Invitrogen Proprietary and Confidential Gene Expression Analysis Technologies High-density Arrays Real-time PCR Number of samples Number of genes >10, >10,000 SAGE Low-density Arrays RPA Northern To consider: Ratio samples / genes, needs for accuracy

4 Invitrogen Proprietary and Confidential Comparison of Quantitative Assays (RNA/DNA): Real-Time PCR Amplicor/TMA NASBA bDNA XPLORE Microarrays RPA Northern Dynamic RangeSensitivity NASBA: nucleic acid seq based amplification bDNA: branched DNA assay Xplore: based on Invader technology TMA: transcription mediated amplification RPA: RNAse protection assay Advantage: Real-Time PCR

5 Invitrogen Proprietary and Confidential Real-time PCR in more details

6 Invitrogen Proprietary and Confidential Phases of amplification Exponential phase Plateau phase

7 Invitrogen Proprietary and Confidential Exponential phase vs. plateau At some time or another, all reactions regardless of initial amount reach the same plateau! –Plateau is not quantitative –Exponential phase is quantitative Cycles Amplicon amount Plateau information is not quantitative

8 Invitrogen Proprietary and Confidential Before the Real-Time era: End-point PCR Examples of semi-quantitative PCR –End point analysis after reduce number of cycles (exponential phase) Not accurate Not sensitive –Competitive PCR Time and reagent consuming GOI (pg) ? PCR condition 95 o C for 2 min # of cycles o C for 15 sec 62 o C for 30 sec 72 o C for 30 sec End Point is at best semi-quantitative

9 Invitrogen Proprietary and Confidential Choices and Decisions

10 Invitrogen Proprietary and Confidential One Step or Two Step RT-PCR?

11 Invitrogen Proprietary and Confidential One Step or Two Step RT-PCR? RNA One Step RT-PCR (One tube) Two Step RT-PCR (Two tubes) Amplicon cDNA GS primers Oligo dT Random Primers (GS Primers) 1 target 1 amplicon X targets X amplicons Target pool

12 Invitrogen Proprietary and Confidential Two-Step RT-PCR Separate conditions for cDNA synthesis & PCR Flexible choice of primers Ideal for quantification of multiple genes from a limited number of RNA samples One-Step RT-PCR Highly defined conditions to support RT and Taq Requires gene specific primer Ideal for quantification of 1 or 2 messages from a large number of RNA samples One Step or Two Step RT-PCR? Perfect for: - Lot of samples - Small amount of targets Perfect for: - Few samples - Large amount of targets Two-step RT-PCR is more convenient and cost effective

13 Invitrogen Proprietary and Confidential Which Reverse Transcriptase?

14 Invitrogen Proprietary and Confidential RNAse H reduced Thermostable ReverseTranscriptase: SuperScript III RT No RNA template degradation > Higher cDNA yield –Improved sensitivity Reduced 5 / 3 bias due to mispriming –Increased reliability Greater percentage of full-length cDNA –complete sequence representation Improved thermostability –high temperature cDNA synthesis –relaxes secondary structure –improved primer specificity Consistent cDNA synthesis efficiency –wide template diversity –wide range of template amount What RT enzyme? Good Reverse transcriptase is essential!

15 Invitrogen Proprietary and Confidential SuperScript III Platinum ® One-Step RT-PCR

16 Invitrogen Proprietary and Confidential Which Polymerase?

17 Invitrogen Proprietary and Confidential Antibody Hot Start - Fast activation - Maximum activity in early cycles Hot Start - Increased specificity - Reduced artifacts (less Primer Dimers) Which Taq polymerase? Platinum® Taq DNA Polymerase Hot Start Taq is a must!

18 Invitrogen Proprietary and Confidential P LATINUM ® Taq automatic hot start PCR Assembly Inactive Taq DNA Polymerase Fully Active Taq DNA Polymerase Temperature Cycling Initial Template Denaturation 94 o C, 30s - 3 min

19 Invitrogen Proprietary and Confidential UDG or no UDG?

20 Invitrogen Proprietary and Confidential Carry-over protection/comparison 20 cycles, ~1,000,000 fold difference DNAAmplicon with dUTP Mix with UDG and dUTP X Amplicon with dUTP Not all UDG kits are built equal!

21 Invitrogen Proprietary and Confidential Which Detection system?

22 Invitrogen Proprietary and Confidential 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 amplicon –Molecular 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 systems –Amplifluor: hairpin primer with fluorophore and quencher –LUX: primer with one fluorophore and no quencher, proprietary to Invitrogen Detection chemistry? Decision, Decision…

23 Invitrogen Proprietary and Confidential Detection chemistry? TaqMan ® Quantiprobe ® SYBR Green I ® Amplifluor Q Q LUX

24 Invitrogen Proprietary and Confidential Which detection system? - Multiplexing - High Specificity - High Sensitivity Probe based or LUX primers -Monoplexing -Cost saving -Fast initial screening Sybr Green I® A detection system for every applications!

25 Invitrogen Proprietary and Confidential LUX TM (Light Upon eXtension) 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 fluorescence LUX Light Upon eXtension!

26 Invitrogen Proprietary and Confidential Quenching effect hairpin secondary structureThe fluorogenic LUX primer has an attached fluorophore at the 3end and a short sequence tail on the 5 end that is complementary to the 3end of the primer. The resulting hairpin secondary structure provides optimal quenching of the attached fluorophore. the terminal dG-dC or dC-dG base pairThe 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.

27 Invitrogen Proprietary and Confidential Competitive 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 (10 7 –10 2 copies). ABI 7700 PCR Efficiency: 93% R 2 :0.998 PCR Efficiency: 92% R 2 :0.999 Quantitect IL6 PCR Efficiency: 94% R 2 :0.996 LUX IL6 TaqMan IL6

28 Invitrogen Proprietary and Confidential Comparison of LUX to TaqMan LUX sensitivity advantages

29 Invitrogen Proprietary and Confidential Melting curve analysis Method: 1.Real-time PCR 2.After amplification: 60°C to 95°C (slow ramp) 3.Fluorescence signal is recorded continuously during the slow temperature ramp 4.Melting curve : Fluorescence signal vs. Temperature 5.Melting curve is converted to melting peaks by plotting –dF/dT vs Temperature LUX Built-in control feature!

30 Invitrogen Proprietary and Confidential Fast control of the PCR product specificity without: - opening the tube and - running a gel Provides accurate real time assessment of quality: - eliminates risk of false positives - reduces risk of contamination Specificity – Melting Curve Advantages LUX versus TaqMan ®

31 Invitrogen Proprietary and Confidential Advantages LUX versus TaqMan ® Alexa 546 TET FAM All detectors Triplex amplification using standard protocol: - 10,000 copies of Gene X are amplified using a LUX primer set labeled with Alexa Fluor ,000 copies of Gene Z are amplified using a LUX primer set labeled with TET copies of Gene Y are amplified using a LUX primer set labeled with FAM Excitation (nm) Emission (nm) FAM JOE TET HEX Alexa Fluor Data from Molecular Probes spectra Easy multiplexing!

32 Invitrogen Proprietary and Confidential Simple Primer Design with the new D-LUX Designer Advantages LUX versus TaqMan ®

33 Invitrogen Proprietary and Confidential 3 Choices to access LUX primers Scientific project D-LUX Designer Self Service Certified LUX primers - HSKG -Virus /Bacteria - Human genes EvoQuest LUX Custom Service Flexibility!

34 Invitrogen Proprietary and Confidential Other convenient formats?

35 Invitrogen Proprietary and Confidential Cells Direct kit No RNA isolation step required. The cDNA synthesis is performed by SuperScript III RNase H - RT. DNase treatment eliminates genomic DNA so you can be confident that results are due to cDNA amplification. Cell Lysis DNase Treatment cDNA Synthesis Application: qPCR All done in 1 tube! From cells to cDNA in ONE TUBE!

36 Invitrogen Proprietary and Confidential From 1 cell to 10,000 cells without NAP! Cells Direct kit

37 Invitrogen Proprietary and Confidential RTS kits Proprietary Polymer Mix Optimized PCR SuperMix Temperature-controlled Lyophilization Packaging Vialing Complete Cycle within 3 days Shelf-life = 1 year Room Temperature Stable

38 Invitrogen Proprietary and Confidential RTS and regular mixes have equal performances RTS kits

39 Invitrogen Proprietary and Confidential What mix for what instrument?

40 Invitrogen Proprietary and Confidential Invitrogen qPCR Reagent Selection Guide SYBR Green DetectionFluorescent Probes/Primers (LUX, TaqMan®, etc.) ABI 7000, 7700, 7900 Roche LightCycler Any Instrument ABI 7000, 7700, 7900 Roche LightCycler Any Instrument DNA or cDNA Platinum SYBR Green qPCR SuperMix-UDG w/ROX Platinum SYBR Green qPCR SuperMix-UDG w/BSA Platinum SYBR Green qPCR SuperMix-UDG Platinum Quantitative PCR SuperMix- UDG w/ROX Platinum Quantitative PCR SuperMix- UDG RNA, 1- Step SuperScript III Platinum SYBR Green One- Step qRT-PCR Kit w/ROX SuperScript III Platinum SYBR Green One- Step qRT-PCR Kit w/BSA SuperScript III Platinum SYBR Green One- Step qRT-PCR Kit SuperScript III Platinum One- Step qRT-PCR Kit w/ROX SuperScript III Platinum One- Step qRT-PCR Kit RNA, 2- Step SuperScript III Platinum SYBR Green Two- Step qRT-PCR Kit w/ROX SuperScript III Platinum SYBR Green Two- Step qRT-PCR Kit w/BSA SuperScript III Platinum SYBR Green Two- Step qRT-PCR Kit SuperScript III Platinum Two- Step qRT-PCR Kit w/ROX SuperScript III Platinum Two- Step qRT-PCR Kit A solution for each problem!

41 Invitrogen Proprietary and Confidential SYBR Green LC qPCR versus Roche qPCR HPRT primers were used with plasmid standards from 1x10 7 to 1x10 1 copies. Invitrogen SYBR Green LC (blue) quantified all 7 logs with a slope of –3.644 and an R-value of No template controls (NTCs) were negative with the SYBR LC kit. Roches 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.

42 Invitrogen Proprietary and Confidential What method of quantitation?

43 Invitrogen Proprietary and Confidential What Method and When

44 Invitrogen Proprietary and Confidential Absolute quantitation

45 Invitrogen Proprietary and Confidential Relative Quantitation: ΔΔCt Method Ct GOI s – Ct norm s = ΔCt Sample Ct GOI c – Ct norm c = ΔCt Calibrator ΔCt Sample – ΔCt Calibrator = ΔΔCt Fold Induction = 2 - ΔΔCt GOI = Gene of Interest Norm = Normalizer (Housekeeping) gene

46 Invitrogen Proprietary and Confidential Where to find more info?

47 Invitrogen Proprietary and Confidential Invitrogen qPCR Central

48 Invitrogen Proprietary and Confidential Invitrogen qPCR Central

49 Invitrogen Proprietary and Confidential Invitrogen multidisplinary qPCR group R&D qPCR group in Carlsbad, CA Enzymologists in Carlsbad, CA Chemists from Molecular Probes in Eugene, OR Custom Primer manufacturing facility in Frederick, MA EvoQuest service group in Carlsbad, CA And more…

50 Invitrogen Proprietary and Confidential The end


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