Presentation on theme: "CFX96 Real-Time PCR Detection System"— Presentation transcript:
1 CFX96 Real-Time PCR Detection System Fast, Friendly, FlexibleDesigned for the Way You WorkRethink PCR
2 Discussion for today• Real time PCR technology• CFX96CFX96 system featuresMethods for optimizationData AnalysisCFX96 software
3 What is Real-Time qPCR?Fluorescence-based detection of amplification products through the use of a DNA-binding dye or hybridization probe.Real-time qPCR is used to quantify input nucleic acid by measuring the number of cycles required to reach a set level of product.In contrast, traditional PCR is used to amplify DNA with end point analysis to distinguish products.What is real time qPCR? It is fluorescence-based detection of amplification products through the use of a DNA-binding dye or probe chemistry.In a qPCR assay one measures the input level of a nucleic acid by determining the number of cycles required to reach a set level of product.This is in contrast to traditional PCR where end-point analysis is performed for product distinction.
4 Limitations of standard PCR Amplification is exponential, but the exponential increase is limited:A linear increase follows exponential phaseEventually plateausTheoreticalReal LifeIn theory, the amount of DNA produced at every cycle should double,Product(T) = (Template0) x 2n(n = # of cycles)Log Target DNACycle #
5 Standard PCR is as endpoint 96 identical reactions will have very different final amounts of fluorescence at endpoint
6 Real-Time PCRThrough the use of fluorescent molecules, real-time PCR has the ability to directly measure the reaction while amplification is taking place.
7 How is quantitative data collected? Real LifeTheoreticalDetectorLog Target DNACycle #
8 Threshold Cycle, CT 96 identical reactions will have almost identical CT values
9 Threshold Cycle, CTThe point at which the fluorescence rises appreciably above backgroundThreshold can be placed anywhere in the exponential (log-linear) phase
10 Threshold SettingAfter baseline subtraction, a threshold line is set empirically or by a statistical calculation at a fluorescence value above background.ThresholdThe first step in analyzing qPCR data is the setting of the Cycle Threshold Line. The threshold line (represented by the dashed line) is a fluorescence value at which standards and samples can be are compared in order to generate quantitative results.The threshold value or position is arbitrary - it can be set to any point in the exponential phase of the reaction. The threshold line is set empirically or by a statistical calculation in the software and it is set at a point where the fluorescence signal rises above background.Log View
11 Mathematical Implications Ideal PCRProductT=(Template0)2nWhere n=Number of CyclesFurther elaboration on the exponential nature of PCR. The mathematic expression that describes the process is shown. Some basic implications are listed.1 CT Difference = 2 fold difference in starting template amount3.3 CT Difference = 10 fold difference in starting template amount
12 Threshold Cycle, CTCorrelates strongly with the starting copy number
13 Threshold Cycle, CT Correlates strongly with the starting copy number 2n = 10 foldn ln 2 = ln 10n = ln10ln 2n = 3.32
14 measurements of starting material. Real-Time PCR: ApplicationsReal-Time reaction monitoring provides information for relative or absolutemeasurements of starting material.Gene Expression StudiesChromatin Immunoprecipitation (ChIP)Methylation Specific PCR (HRM)Microarray ValidationTransgenic AnalysisGMO TestingViral/Bacterial Load StudiesAllelic Discrimination/SNP (HRM)
15 From CT values, we can determine the initial copy number
16 Chemistries used in real time PCR Intercalation DyesHybridization Probes
17 Intercalation (DNA binding) dyes DNA binding dyes are inexpensive compared to hybridization probes.EtBr is 25 times more fluorescent when bound to dsDNASYBR Green I is 125 times more fluorescent brightly bound to dsDNA
18 Intercalation Dyes:SYBR Green IlllTaqID5’3’5’Taq5’3’ll
19 Advantages Disadvantages SYBR Green I Experiment only requires primers Potential contribution to fluorescence from non-specific products (primer-dimers)No multiplexingWhen used for real-time qPCR, SGI has the following advantages over probe based chemistries.It allows for easy experimental design since you only need the forward and reverse amplification primers. No additional probes are required. So, if you already have a PCR assay for a particular target, you may be able to convert it to a SYBR Green I real time qPCR assay using by following some of the optimization guidelines that will be discussed later in this presentation.SGI does have some disadvantages. Since it is an indiscriminate dsDNA binding dye, there could be potential contribution to overall fluorescence from non-specific products (primer-dimers). Again, optimization guidelines that are useful to eliminate problems with primer-dimers will be discussed later in this presentation.In addition, multiplexing cannot be performed when using SGI.
20 Hybridization Probes Currently, hybridization probe strategies fall into three main categories:Cleavage-based assayTaqManä AssaysLocked nucleic acids (LNA)Displaceable probe assaysmolecular beaconsDual-oligo FRET probesProbes incorporated directly into the primersAmplifluor & Scorpions
23 TaqMan Advantages Disadvantages Target specific fluorescence MultiplexingDisadvantagesHigh initial costAssay design not trivialCompared to SYBR Green I chemistry, TaqMan probes have the advantages of target specific fluorescence, and the ability to be used in multiplexing reactions since the probes specific to different targets can be labeled with different fluorophores.The disadvantages of TaqMan probes are that there is a high initial cost and a more difficult assay design process due to the need for a hybridization probe as well as amplification primers. Optimization guidelines and a discussion of Beacon Designer software that can be used to design TaqMan assays will be discussed later in this presentation.
24 Real Time PCR Technology: -Enables detection and quantification of sample-Extremely sensitive-Can be used in various applications (gene expression,allelic discrimination, pathogen detection)Compared to SYBR Green I chemistry, TaqMan probes have the advantages of target specific fluorescence, and the ability to be used in multiplexing reactions since the probes specific to different targets can be labeled with different fluorophores.The disadvantages of TaqMan probes are that there is a high initial cost and a more difficult assay design process due to the need for a hybridization probe as well as amplification primers. Optimization guidelines and a discussion of Beacon Designer software that can be used to design TaqMan assays will be discussed later in this presentation.Questions?
26 Unsurpassed Thermal Cycling CFX96 builds on the precise thermal control of the C1000Maintain temperature uniformity while ramping10 second settling - the time it takes all wells to reach temperatureMax ramp rate5oC/secAverage ramp rate3.3oC/secTemp Accuracy± 0.2oCTemp Uniformity± 0.4oC in 10 secTemp Range0-100oCThe power and precision of any real time system really depends on the thermacycler. The CFX96 builds on the precise thermal control of the C1000 cycler, aintain uniformity even while ramping10 second settling - the time it takes all wells to reach temperature - is unsurpassed in the market
27 Fast block architecture Patented Block DesignFast block architectureTo achieve faster ramp rates, you must reduce block mass.This ingenious design removes mass without sacrificing rigidity (describe).Note the this design is patent pending….Mass-reduced sample block** Patented by Bio-Rad
28 Time to Temperature1000-Series Thermal Cycler Time to TemperatureYou can also be assured that your block maintains temperature unifomrity across all the wells. In this experiment 15 temperature probes monitored the temperature of wells on the perimeter of the bloack as well as inside,So you can be assured the ramping in the block is uniform. This feature is going to allow you to achieve fast PCR runs.Probe LocationUniform ramping + shorter settling times = Faster PCR
29 CFX96 Optical Technology Scanning optics shuttle6 filtered LEDs for excitation6 filtered photodiodes for detectionMultiplex up to 5 targetsIndependently illuminate and detect fluorescence in each channel during scan
30 Next Generation Optical Technology: CFX96 uses a scanning shuttle6 filtered LEDs for excitation6 filtered photodiodes for detectionLEDs fire sequentiallyMultiplex up to 5 targetsAll dyes excited near their maximaFixed optical path for all wellsNo cross talkData is collected for all wells in all channels
31 Optical Technology provides hassle free maintenance LEDs are long lastingFactory calibrated. Does not require recalibrationNo need for Passive Reference (Rox)Data is always acquired from all wells in all channels>100/well/scanLaser Homing of shuttle at every scanWorry free performance over the lifetime of the instrument, LEDs do not burn out and they exhibit limited light degradationNo sacrificing a channel to normalize for positional bias with ROX, fluorescence is measured at the same distance for each wellData can be collected in all channels therefore you never lose data from an experimental run.
32 Multiple Fast Scan Modes Channel(s)Scan Time (sec)All Channels1-512SYBR/FAM Only13FRET6
33 Excellent Uniformity at 10l Fast ScanAll ChannelsAve Ct = ± 0.12Ave Ct = ± 0.12A full 96 well plate was used for these experiments.
34 Flexibility to use 6 Filter Sets ChannelExcitation (nm)Detection (nm)Calibrated Fluorophores1FAM™, SYBR Green I™2VIC®, HEX™, TET™, Cal Gold 540™3ROX™, TEXAS RED®, Cal Red 610™4CY5, Quasar 670™5Quasar 705™6Accommodates FRET ChemistryNo need to recalibrate, ever. Reliable. Stable. Long life. Hassle free.
35 Unsurpassed Dye Separation Achieve sensitive multiplexing by maximal excitation and detection of dyes12345FamHexTxRedCy5Q705100002000030000400005000060000ChannelSignalFluorophoreDiscrete Excitation and Detection of Dyes Even without color separating fluorescence between channels, there is almost no dye cross talk between channelsUsing filtered LEDs and photodiodes ensure that each dye is excited close to it’s maximal wavelength and detected close to it’s optimal emission spectra. This ensures sensitivity and reproducibility in single dye assays a s well as multiplexing experiments.Shown here are the detection of each of these dyes multiplexed together. As you can see there is excellent separation of the dyes with no crosstalk between dyes.
36 Excellent Uniformity at 10l Ave Ct = ± 0.11Max-Min =0.52Ave Ct = ± 0.11Max-Min =0.61HexTexas RedAgain, excellent uniformity in all wells and all dye channels.
37 Excellent Uniformity at 10l in all channels Ave Ct = ± 0.12Max-Min =0.62Ave Ct = ± 0.07Max-Min =0.37Cy5Quasar 705It doesn’t matter what dye you choose the uniformity of data is still the same. This is an example of being able to excite the dyes near their maxima and detect them near their emissions maxima. You can be confident no matter what dye you use even in multiplexing.
38 CFX96 features for Reaction Optimization Melt Curve –MIQE GuidelinesThermal GradientFast RT-PCRData Analysis
39 Melting temperature (Tm) of dsDNA Melt Curve AnalysisPrinciple:After PCR amplification, the temperature is increased, causing the dsDNA to melt and release SGI, resulting in a decrease in fluorescenceAnalogous to agarose gel analysis except Tm is used to distinguish productsMelting temperature (Tm) of dsDNATemperature at which half the DNA is double stranded and half is single strandedDepends on nucleotide content and lengthWhen using SYBR Green I for real-time quantification, one can perform post amplification melting curve analysis to distinguish reaction products and analyze reaction specificity, eliminating the need for agarose gel analysis of reaction products.It is complementary to running a gel, except Tm is used to distinguish the reaction products.The principle of the melting curve is that the temperature is ramped from a low temp (all sequences annealed) to a high temp to cause strand dissociation. As the dsDNA melts, SGI is released and a decrease in fluorescence is observed. The melting temperature is determined predominantly by the nucleotide content. In general, amplicons with high GC content require a higher temperature to melt and will subsequently have a higher Tm compared to AT-rich amplicons.
40 Melt Curve AnalysisAfter real-time PCR amplification, a melt curve is performed in presence of a DNA binding “saturation dye”Melting temperature (Tm)DNA is half double and half single-strandedDepends on nucleotide content and lengthTmDouble Stranded DNASingle Stranded
41 Melt Curve AnalysisEndpoint analysis to determine the melting temperature (Tm) of PCR products.
43 Thermal GradientUsed for one-step reaction-temperature optimization for PCR reaction specificity and efficiency.Up to 25oC gradient range programmable across block.“Dynamic Ramping” - cycler maintains the same hold time for each temperature.
44 Thermal Gradient SYBR Green I chemistry annealing temperature primer concentrationUse temperature gradient featureLook for lowest Ct valueTemperaturegradientDilution series of primer [ ]SYBR Green I chemistry
45 Optimization of Annealing Temperature for Best Results Annealing temperature is critical forSpecificityReproducibilityPCR Reaction EfficiencySensitivityReliable data67oC62oC56oCEfficiency = 68%Efficiency = 99%Efficiency = 98%Serial dilutions8 temps from 55oC to 68oC62oC is optimal-low Cts and highest reactionefficiencyThe thermal gradient in the CFX allows you to quickly and easily optimize your results for the highest sensitivity and reproducibility possible. Optimization is an empirical process dependent on our specific reaction conditions.This slide demonstrates that the assay shown here is optimal at 62C. Look at the PCR efficiencies in the other temperatures. Also not the replicates at 67C. 62C will produce more sensitive reproducible data in this assayDynamic ramping means that the your samples reach the set temperature at the same time to eliminate time as a reaction variable. If you are optimizing your reactions using a thermal gradient then you want the reactions all to incubate at the set times. In other temperature gradients on the market the ramp rate is constant meaning that the incubation times for each temperature will be different.The temperature gradient is set from hottest in row A (rear of the block) to coolest in row H (front of the block).
46 Fast PCR: 3-step PCR vs 2-step PCR DenaturationAnnealingExtension
48 SsoFast Eva Green Supermix: Sso7d-fusion Protein Technology Sso7d from Sulfolobus solfataricus7kD, 63 aa.Thermostable (Tm >90°C)No sequence preferenceBinds to dsDNA (3-6 bp/protein molecule)Monomeric
49 SsoFast Eva Green Supermix: EvaGreen Dye EvaGreen dye is similar to SYBR® Green IVery low PCR inhibitionIncreased sensitivityFast qPCR
50 Data Analysis:Basic delta CtDelta-delta CtPfaffl delta-delta Ct
51 (no normalization to reference gene) Calculating for relative quantitationBasic delta Ct method:(no normalization to reference gene)Primer set #2Tissue #1:22Tissue #2:24Delta Ct:24-22 = 2Fold induction =22 = 4
52 (assumes same efficiencies for each primer set) Calculating for relative quantitationDelta-delta Ct method:(assumes same efficiencies for each primer set)Reference Primer setGOI Primer setTissue #1:2122Tissue #2:2024Delta Ct:22-21 = 11st DeltaDelta Ct:24-20 = 42nd DeltaDelta Ct:4-1 = 3Fold induction =23 = 8
53 Ct SQ Calculating for relative quantitation Problems of delta-delta Ct method:Ct242290%SQ
54 Ct SQ Calculating for relative quantitation Problems of delta-delta Ct method:Ct242290%100%SQ
55 Calculating for relative quantitation Problem with the CTSlopes are not parallelCt242290%100%Starting quantity
57 Calculating for relative quantitation Pfaffl method:(efficiencies are normalized)Primer set #1ReferencePrimer set #2 GOITissue #1:2122Tissue #2:2024(From Standard curve)Efficiency:90% = 1.9100% = 2Delta Ct:20-21 = -124-22 = 22target deltaCt target (24-22 = 2)47.57.5Fold induction ===0.530.531.9reference deltaCt reference (20-21 = -1)
58 Comparison of methods for relative quantitation calculations Basic delta Ct method: (no reference gene)Fold induction : 4Delta-delta Ct method: (reference gene)Fold induction : 8Ideal for primer pairs with an E ≥ 90% AND large fold changes in expression (10 fold or more)Pfaffl method: (reference gene and efficiency)Fold induction : 7.5
59 Relative Gene Expression Analysis What to Use as StandardsPlasmid DNAPCR ProductSpiked sample (with plasmid or PCR product)Positive cDNA control but unknown concentration (dilution)
60 There are no true “House keeping” genes Vandesompele MethodThere are no true “House keeping” genesUses more than 1 reference gene (3 is recommended) and takes the geometric mean to normalize fold expressionUsing a single reference gene leads to erroneous normalization up to 3.0-fold and 6.4-fold in 25% and 10% of the cases, respectively, with sporadic values above 20-foldgeNorm site:geNorm is a popular algorithm to determine the most stable reference (housekeeping) genes from a set of tested candidate reference genes in a given sample panel
61 Bio-Rad: Experts in Real-time PCR Bio-Rad’s Innovation in Real-time PCR continues with the CFX96We can help you achieve success at every step of your researchIn-house ScientistsField Application ScientistsField ServiceTechnical SupportField Sales RepresentativesRethink PCR
62 CFX96 Real-Time PCR System Questions?Thank you for joining us!