Real-Time quantitative PCR:Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005
Gene Expression Analysis Technologies >10,000 Real-time PCR 100 Low-density Arrays Number of samples 10 High-density Arrays RPA Northern SAGE 1 2-10 100-1000 >10,000 Number of genes To consider: Ratio samples / genes, needs for accuracy
Comparison of Quantitative Assays (RNA/DNA): Sensitivity Dynamic Range Real-Time PCR Amplicor/TMA NASBA bDNA XPLORE Microarrays RPA Northern 100 101 102 103 104 105 106 107 108 108 107 106 105 104 103 102 101 100 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
Exponential phase Plateau phase Phases of amplification
Amplicon amount Cycles 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 Plateau information is not quantitative
? 0 0.1 1 10 GOI (pg) PCR condition 95oC for 2 min # of cycles 30 95oC for 15 sec 62oC for 30 sec 72oC for 30 sec 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 End Point is at best semi-quantitative
One Step or Two Step RT-PCR? One Step RT-PCR (One tube) Two Step RT-PCR (Two tubes) RNA 1 target X targets Oligo dT Random Primers (GS Primers) GS primers cDNA Target pool 1 amplicon Amplicon Amplicon X amplicons
One Step or Two Step RT-PCR? • 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 • 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 • 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
RNAse H reduced Thermostable ReverseTranscriptase:SuperScript III ™ RT What RT enzyme? • 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 Good Reverse transcriptase is essential!
Which Taq polymerase? Hot Start - Increased specificity - Reduced artifacts (less Primer Dimers) Antibody Hot Start - Fast activation - Maximum activity in early cycles Platinum® Taq DNA Polymerase Hot Start Taq is a must!
PLATINUM®Taq automatic hot start Initial Template Denaturation PCR Assembly Temperature Cycling 94oC, 30s - 3 min Inactive Taq DNA Polymerase Fully Active Taq DNA Polymerase
Carry-over protection/comparison Mix with UDG and dUTP DNA Amplicon with dUTP X Amplicon with dUTP 20 cycles, ~1,000,000 fold difference Not all UDG kits are built equal!
Detection chemistry? • 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 Decision, Decision…
SYBR Green I® TaqMan® Quantiprobe® Q Q Amplifluor™ LUX™ Detection chemistry?
Which detection system? • Monoplexing • Cost saving • Fast initial screening • Sybr Green I® • - Multiplexing • - High Specificity • - High Sensitivity • Probe based • or • LUX™ primers A detection system for every applications!
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 fluorescence Light Upon eXtension!
Quenching effect • The 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.
TaqMan IL6 Quantitect IL6 LUX IL6 PCR Efficiency: 92% R2:0.999 PCR Efficiency: 94% R2:0.996 PCR Efficiency: 93% R2:0.998 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 (107 –102 copies). ABI 7700
Comparison of LUX™ to TaqMan LUX™ sensitivity advantages
Melting curve analysis Method: • Real-time PCR • After amplification: 60°C to 95°C (slow ramp) • Fluorescence signal is recorded continuously during the slow temperature ramp • Melting curve : Fluorescence signal vs. Temperature • Melting curve is converted to melting peaks by plotting –dF/dT vs Temperature LUX™ Built-in control feature!
Advantages LUX™ versus TaqMan® Specificity – Melting Curve • 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
Alexa 546 All detectors TET FAM Advantages LUX™ versus TaqMan® Data from Molecular Probes spectra Triplex 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 FAM Easy multiplexing!
Advantages LUX™ versus TaqMan® Simple Primer Design with the new D-LUX™ Designer www.invitrogen.com/dluxdesigner
3 Choices to access LUX™ primers Scientific project D-LUX™ Designer Self Service EvoQuest™ LUX™ Custom Service • Certified LUX™ primers • - HSKG • Virus /Bacteria • Human genes Flexibility!
Cells Direct kit Cell Lysis No RNA isolation step required. DNase treatment eliminates genomic DNA so you can be confident that results are due to cDNA amplification. DNase Treatment All done in 1 tube! The cDNA synthesis is performed by SuperScript™ III RNase H- RT. cDNA Synthesis Application: qPCR From cells to cDNA in ONE TUBE!
Cells Direct kit From 1 cell to 10,000 cells without NAP!
RTS kits Optimized PCR SuperMix Vialing Proprietary Polymer Mix Temperature-controlled Lyophilization Packaging Shelf-life = 1 year Complete Cycle within 3 days Room Temperature Stable
Standard Curve RTS one step 40 y = -3.548(x) + 37.1 2 R = 0.998 35 30 Cycle Threshold 25 20 15 10 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Starting Concentration Standard Curve aqueous (SuperScript III one step) 40 y = -3.505(x) + 36.8 2 R = 0.999 35 30 Cycle Threshold 25 20 15 10 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Starting Concentration RTS kits RTS and regular mixes have equal performances
Invitrogen qPCR Reagent Selection Guide A solution for each problem!
SYBR Green LC qPCR versus Roche qPCR HPRT 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 -1.00. 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 -1.00. The NTCs along with the last two dilutions showed contamination.
What Method and When http://www.gene-quantification.de/strategy.html
CtGOIs – Ctnorms = ΔCtSample CtGOIc – Ctnormc = ΔCtCalibrator GOI = Gene of Interest Norm = Normalizer (Housekeeping) gene ΔCtSample– ΔCtCalibrator=ΔΔCt Fold Induction = 2- ΔΔCt Relative Quantitation: ΔΔCt Method
Invitrogen qPCR Central www.invitrogen.com/qpcr
Invitrogen qPCR Central www.invitrogen.com/qpcr
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…
The end www.invitrogen.com/qpcr