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Version 1.0 CFX96 Real-Time PCR Detection System Rethink PCR Fast, Friendly, Flexible Designed for the Way You Work.

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Presentation on theme: "Version 1.0 CFX96 Real-Time PCR Detection System Rethink PCR Fast, Friendly, Flexible Designed for the Way You Work."— Presentation transcript:

1 Version 1.0 CFX96 Real-Time PCR Detection System Rethink PCR Fast, Friendly, Flexible Designed for the Way You Work

2 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Discussion for today Real time PCR technology CFX96 CFX96 system features –Methods for optimization –Data Analysis CFX96 software

3 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 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.

4 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 A linear increase follows exponential phase Eventually plateaus Cycle # Theoretical Real Life Log Target DNA Limitations of standard PCR Amplification is exponential, but the exponential increase is limited: In theory, the amount of DNA produced at every cycle should double, Product(T) = (Template0) x 2 n (n = # of cycles)

5 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Standard PCR is as endpoint 96 identical reactions will have very different final amounts of fluorescence at endpoint

6 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Real-Time PCR Through the use of fluorescent molecules, real-time PCR has the ability to directly measure the reaction while amplification is taking place.

7 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 How is quantitative data collected? Cycle # Theoretical Real Life Log Target DNA Detector

8 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 96 identical reactions will have almost identical C T values Threshold Cycle, C T

9 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Threshold Cycle, C T The point at which the fluorescence rises appreciably above background Threshold can be placed anywhere in the exponential (log-linear) phase

10 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Threshold Setting After baseline subtraction, a threshold line is set empirically or by a statistical calculation at a fluorescence value above background. Threshold Log View

11 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 1 C T Difference = 2 fold difference in starting template amount 3.3 C T Difference = 10 fold difference in starting template amount Product T =(Template 0 )2 n Where n=Number of Cycles Mathematical Implications Ideal PCR

12 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Threshold Cycle, C T Correlates strongly with the starting copy number

13 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Threshold Cycle, C T Correlates strongly with the starting copy number 2 n = 10 fold n ln 2 = ln 10 n = ln10 ln 2 n = 3.32

14 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Real-Time PCR: Applications Real-Time reaction monitoring provides information for relative or absolute measurements of starting material. Gene Expression Studies Chromatin Immunoprecipitation (ChIP) Methylation Specific PCR (HRM) Microarray Validation Transgenic Analysis GMO Testing Viral/Bacterial Load Studies Allelic Discrimination/SNP (HRM)

15 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 From C T values, we can determine the initial copy number

16 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Intercalation Dyes Hybridization Probes Chemistries used in real time PCR

17 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Intercalation (DNA binding) dyes n DNA binding dyes are inexpensive compared to hybridization probes. n EtBr is 25 times more fluorescent when bound to dsDNA n SYBR Green I is 125 times more fluorescent brightly bound to dsDNA

18 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Intercalation Dyes: SYBR Green I 3’ 5’ 3’ Taq 5’ ID

19 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 SYBR Green I Advantages –Experiment only requires primers Disadvantages –Potential contribution to fluorescence from non- specific products (primer-dimers) –No multiplexing

20 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Hybridization Probes Cleavage-based assay TaqMan  Assays Locked nucleic acids (LNA) Displaceable probe assays molecular beacons Dual-oligo FRET probes Probes incorporated directly into the primers Amplifluor & Scorpions Currently, hybridization probe strategies fall into three main categories:

21 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 5’ 3’ d. NTPs Thermal Stable DNA Polymerase Primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Add iQ Supermix, Hybridization Probe and sample Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Annealing Taq 5’ 3’ R Q Probe 5’ 3’ R Q Cleavage-based assay: TaqMan TM

22 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 5’ 3’ 5’ 3’ 5’ 3’ R Q 5’ 3’ Taq 3’ QR 5’ 3’ Q Taq R 5’ 3’ Q Taq R 3’ 5’ Extension Step 5’ 3’ Q Taq R 5’ Cleavage-based assay: TaqMan TM

23 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 TaqMan Advantages –Target specific fluorescence –Multiplexing Disadvantages –High initial cost –Assay design not trivial

24 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Real Time PCR Technology: Questions? Real-Time PCR: -Enables detection and quantification of sample -Extremely sensitive -Can be used in various applications (gene expression, allelic discrimination, pathogen detection)

25 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Modular thermal cycler platform, includes C1000 thermal cycler chassis, CFX96 optical reaction module, CFX Manager software CFX 96 Real-Time PCR Detection System

26 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Unsurpassed Thermal Cycling CFX96 builds on the precise thermal control of the C1000 –Maintain temperature uniformity while ramping –10 second settling - the time it takes all wells to reach temperature Max ramp rate5 o C/sec Average ramp rate3.3 o C/sec Temp Accuracy± 0.2 o C Temp Uniformity± 0.4 o C in 10 sec Temp Range0-100 o C

27 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Mass-reduced sample block* Patented Block Design * Patented by Bio-Rad Fast block architecture

28 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Time to Temperature Probe Location 1000-Series Thermal Cycler Time to Temperature Uniform ramping + shorter settling times = Faster PCR

29 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 CFX96 Optical Technology Scanning optics shuttle 6 filtered LEDs for excitation 6 filtered photodiodes for detection Multiplex up to 5 targets Independently illuminate and detect fluorescence in each channel during scan

30 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Next Generation Optical Technology: CFX96 uses a scanning shuttle –6 filtered LEDs for excitation –6 filtered photodiodes for detection –LEDs fire sequentially Multiplex up to 5 targets All dyes excited near their maxima Fixed optical path for all wells No cross talk Data is collected for all wells in all channels

31 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Optical Technology provides hassle free maintenance LEDs are long lasting Factory calibrated. Does not require recalibration No need for Passive Reference (Rox) Data is always acquired from all wells in all channels >100/well/scan Laser Homing of shuttle at every scan

32 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Multiple Fast Scan Modes ModeChannel(s)Scan Time (sec) All Channels1-512 SYBR/FAM Only13 FRET63

33 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Excellent Uniformity at 10  l Fast ScanAll Channels Ave Ct = 19.29 ± 0.12 Ave Ct = 19.81 ± 0.12

34 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Flexibility to use 6 Filter Sets ChannelExcitation (nm)Detection (nm)Calibrated Fluorophores 1450-490515-530FAM™, SYBR Green I™ 2515-535560-580VIC ®, HEX™, TET™, Cal Gold 540™ 3560-590610-650ROX™, TEXAS RED ®, Cal Red 610™ 4620-650675-690CY5, Quasar 670™ 5672-684705-730Quasar 705™ 6450-490560-580Accommodates FRET Chemistry No need to recalibrate, ever. Reliable. Stable. Long life. Hassle free.

35 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Unsurpassed Dye Separation Achieve sensitive multiplexing by maximal excitation and detection of dyes

36 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Ave Ct = 19.67 ± 0.11 Max-Min =0.52 Ave Ct = 19.21 ± 0.11 Max-Min =0.61 HexTexas Red Excellent Uniformity at 10  l

37 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Ave Ct = 19.96 ± 0.12 Max-Min =0.62 Ave Ct = 19.27 ± 0.07 Max-Min =0.37 Cy5Quasar 705 Excellent Uniformity at 10  l in all channels

38 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 CFX96 features for Reaction Optimization Melt Curve –MIQE Guidelines Thermal Gradient Fast RT-PCR Data Analysis

39 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Melt Curve Analysis Principle: –After PCR amplification, the temperature is increased, causing the dsDNA to melt and release SGI, resulting in a decrease in fluorescence Analogous to agarose gel analysis except Tm is used to distinguish products Melting temperature (Tm) of dsDNA –Temperature at which half the DNA is double stranded and half is single stranded –Depends on nucleotide content and length

40 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Melt Curve Analysis After 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-stranded –Depends on nucleotide content and length Tm Double Stranded DNA Single Stranded

41 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Endpoint analysis to determine the melting temperature (Tm) of PCR products. Melt Curve Analysis

42 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Melt Curve Analysis: Primer Dimer

43 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Used for one-step reaction- temperature optimization for PCR reaction specificity and efficiency. Up to 25 o C gradient range programmable across block. “Dynamic Ramping” - cycler maintains the same hold time for each temperature. Thermal Gradient

44 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 1)annealing temperature 2)primer concentration Use temperature gradient feature Look for lowest Ct value Temperature gradient Dilution series of primer [ ] SYBR Green I chemistry Thermal Gradient

45 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Optimization of Annealing Temperature for Best Results Serial dilutions 8 temps from 55 o C to 68 o C 62 o C is optimal -low Cts and highest reaction efficiency 67 o C 62 o C 56 o C Efficiency = 68% Efficiency = 99% Efficiency = 98% Annealing temperature is critical for Specificity Reproducibility PCR Reaction Efficiency Sensitivity Reliable data

46 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 AnnealingExtensionDenaturation 95ºC 58ºC 72ºC Fast PCR: 3-step PCR vs 2-step PCR

47 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Quality Assays – SsoFast Eva Green Supermix

48 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 SsoFast Eva Green Supermix: Sso7d-fusion Protein Technology Sso7d from Sulfolobus solfataricus –7kD, 63 aa. –Thermostable (Tm >90°C) –No sequence preference –Binds to dsDNA (3-6 bp/protein molecule) –Monomeric

49 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 EvaGreen dye is similar to SYBR® Green I Very low PCR inhibition Increased sensitivity Fast qPCR SsoFast Eva Green Supermix: EvaGreen Dye

50 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Basic delta Ct Delta-delta Ct Pfaffl delta-delta Ct Data Analysis:

51 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Calculating for relative quantitation Basic delta Ct method: (no normalization to reference gene) Primer set #2 Tissue #2: Tissue #1:22 24 Delta Ct:24-22 = 2 Fold induction = 2 2 = 4

52 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Calculating for relative quantitation Delta-delta Ct method: (assumes same efficiencies for each primer set) Reference Primer setGOI Primer set 21 Tissue #2: Tissue #1: 20 22 24 Delta Ct: 22-21 = 1 24-20 = 4 1 st Delta Delta Ct:4-1 = 32 nd Delta Fold induction =2 3 = 8

53 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Calculating for relative quantitation Problems of delta-delta Ct method: CtCt SQ 90% 24 22

54 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Calculating for relative quantitation Problems of delta-delta Ct method: CtCt SQ 90% 24 22 100%

55 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 CtCt 90% 24 22 100% Starting quantity Problem with the  CT Slopes are not parallel Calculating for relative quantitation

56 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Pfaffl method: (Pfaffl, 2001; Nucleic Acid Research) Efficiency target deltaCt target (control-sample) Fold induction = Efficiency reference deltaCt reference (control-sample) Efficiency = 10 -1/slope Calculating for relative quantitation Pfaffl method: (Pfaffl, 2001; Nucleic Acid Research) Efficiency target deltaCt target (control-sample) Fold induction = Efficiency reference deltaCt reference (control-sample)

57 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Pfaffl method: (efficiencies are normalized) Primer set #1ReferencePrimer set #2 GOI 21 Tissue #2: Tissue #1: 20 22 24 90% = 1.9 Delta Ct: Efficiency: 20-21 = -1 100% = 2 24-22 = 2 2 target deltaCt target (24-22 = 2) Fold induction = 1.9 reference deltaCt reference (20-21 = -1) = 4 0.53 = 7.5 0.53 7.5 (From Standard curve) Calculating for relative quantitation

58 www.bio-rad.com/pcr AMPLIFICATION Version 1.0  Basic delta Ct method: (no reference gene)  Fold induction : 4  Delta-delta Ct method: (reference gene)  Fold induction : 8  Ideal 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 Comparison of methods for relative quantitation calculations

59 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Relative Gene Expression Analysis Plasmid DNA PCR Product Spiked sample (with plasmid or PCR product) Positive cDNA control but unknown concentration (dilution) What to Use as Standards

60 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Vandesompele Method There are no true “House keeping” genes Uses more than 1 reference gene (3 is recommended) and takes the geometric mean to normalize fold expression Using 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-fold geNorm site: http://medgen.ugen.be/~jvdesomp/genorm/http://medgen.ugen.be/~jvdesomp/genorm/ –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 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 Bio-Rad: Experts in Real-time PCR Bio-Rad’s Innovation in Real-time PCR continues with the CFX96 We can help you achieve success at every step of your research –In-house Scientists –Field Application Scientists –Field Service –Technical Support –Field Sales Representatives –www.bio-rad.com/genomics Rethink PCR

62 www.bio-rad.com/pcr AMPLIFICATION Version 1.0 CFX96 Real-Time PCR System Questions? Thank you for joining us!


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