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

Why using real-time PCR?

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

Real-time PCR in more details

Phases of amplification Exponential phase Plateau phase

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

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) 0.1 1 10 ? PCR condition 95oC for 2 min # of cycles 30 95oC for 15 sec 62oC for 30 sec 72oC for 30 sec End Point is at best semi-quantitative

Choices and Decisions

One Step or Two Step RT-PCR?

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

Two-step RT-PCR is more convenient and cost effective 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

Which Reverse Transcriptase?

Good Reverse transcriptase is essential! 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 RNAse H reduced Thermostable ReverseTranscriptase: SuperScript III ™ RT Good Reverse transcriptase is essential!

SuperScript™ III Platinum® One-Step RT-PCR

Which Polymerase?

Platinum® Taq DNA Polymerase 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

UDG or no UDG?

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

Which Detection system?

Detection 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 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…

Detection chemistry? SYBR Green I® TaqMan® Amplifluor™ LUX™ Quantiprobe® Amplifluor™ Q LUX™

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!

Light 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 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.

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 PCR Efficiency: 93% R2:0.998 PCR Efficiency: 92% R2:0.999 Quantitect IL6 PCR Efficiency: 94% R2:0.996 LUX IL6 TaqMan IL6

Comparison of LUX™ to TaqMan LUX™ sensitivity advantages

LUX™ Built-in control feature! 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

Advantages LUX™ versus TaqMan® Alexa 546 TET FAM All detectors Excitation (nm) Emission FAM 492 517 JOE 520 548 TET 521 536 HEX 533 550 Alexa Fluor 546 554 570 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!

Other convenient formats?

From cells to cDNA in ONE TUBE! 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!

From 1 cell to 10,000 cells without NAP! Cells Direct kit From 1 cell to 10,000 cells without NAP!

Room Temperature Stable 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

RTS and regular mixes have equal performances RTS kits Standard Curve RTS one step y = -3.548(x) + 37.1 R 2 = 0.998 10 15 20 25 30 35 40 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Starting Concentration Cycle Threshold Standard Curve aqueous (SuperScript III one step) y = -3.505(x) + 36.8 R 2 = 0.999 10 15 20 25 30 35 40 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Starting Concentration Cycle Threshold RTS and regular mixes have equal performances

What mix for what instrument?

Invitrogen qPCR Reagent Selection Guide SYBR Green Detection Fluorescent Probes/Primers (LUX™, TaqMan®, etc.) 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 We will test AM’s on the most suitable kit for the two customer examples outlined earlier. A solution for each problem!

SYBR Green LC qPCR versus Roche qPCR Title of slide is set as Arial, Bold, Italic, size 24 Takeaway 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 Arial Only italicize Titles and Text Boxes – use Title Case Don’t do all caps for titles or column headings …use Title Case No gray fill in objects where you have black type ... doesn’t copy well No shadowing … on anything Keep graphics very simple … 2D vs. 3D … keep colors compatible throughout presentation Keep text within the boundaries so can be viewed well when on a large screen 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 of quantitation?

What Method and When http://www.gene-quantification.de/strategy.html

Absolute quantitation

Relative Quantitation: ΔΔCt Method CtGOIs – Ctnorms = ΔCtSample CtGOIc – Ctnormc = ΔCtCalibrator ΔCtSample – ΔCtCalibrator = ΔΔCt Fold Induction = 2- ΔΔCt GOI = Gene of Interest Norm = Normalizer (Housekeeping) gene

Where to find more info?

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