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

2. Discussion for today
• Real time PCR technology
• CFX96
CFX96 system features
Methods for optimization
Data Analysis
CFX96 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 phase
Eventually plateaus
Theoretical
Real Life
In theory, the amount of DNA produced at every cycle should double,
Product(T) = (Template0) x 2n
(n = # of cycles)
Log Target DNA
Cycle #

5. Standard PCR is as endpoint
96 identical reactions will have very different final amounts of fluorescence at endpoint

6. 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. How is quantitative data collected?
Real Life
Theoretical
Detector
Log Target DNA
Cycle #

8. Threshold Cycle, CT 96 identical reactions will have
almost identical CT values

9. Threshold Cycle, CT
The point at which the fluorescence rises appreciably above background
Threshold can be placed anywhere in the exponential (log-linear) phase

10. Threshold Setting
After baseline subtraction, a threshold line is set empirically or by a statistical calculation at a fluorescence value above background.
Threshold
The 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 PCR
ProductT=(Template0)2n
Where n=Number of Cycles
Further 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 amount
3.3 CT Difference = 10 fold difference in starting template amount

12. Threshold Cycle, CT
Correlates strongly with the starting copy number

13. Threshold Cycle, CT Correlates strongly with the starting copy number
2n = 10 fold
n ln 2 = ln 10
n = ln10
ln 2
n = 3.32

14. measurements of starting material.
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. From CT values, we can determine
the initial copy number

16. Chemistries used in real time PCR
Intercalation Dyes
Hybridization Probes

17. Intercalation (DNA binding) dyes
DNA binding dyes are inexpensive compared to hybridization probes.
EtBr is 25 times more fluorescent when bound to dsDNA
SYBR Green I is 125 times more fluorescent brightly bound to dsDNA

18. Intercalation Dyes:
SYBR Green I l l l
Taq ID 5’ 3’ 5’
Taq 5’ 3’ l l

19. Advantages Disadvantages SYBR Green I Experiment only requires primers
Potential contribution to fluorescence from non-specific products (primer-dimers)
No multiplexing
When 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 assay
TaqManä Assays
Locked nucleic acids (LNA)
Displaceable probe assays
molecular beacons
Dual-oligo FRET probes
Probes incorporated directly into the primers
Amplifluor & Scorpions

21. Cleavage-based assay: TaqManTM5’ 3’ 5’ 3’
Add iQ Supermix,
Hybridization Probe
and sample
d.NTPs
Primers
Thermal Stable
DNA Polymerase 5’ 3’ R Q
Probe
Denaturation 5’ 3’ 5’ 3’
Taq 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ l 5’ 3’ R Q
Annealing

22. Cleavage-based assay: TaqManTM5’ 3’ R Q 5’ 3’ 5’ 3’ R
Taq
Extension Step 3’ Q 5’ 5’ 3’ R
Taq 3’ Q 5’ 5’ 3’
Taq R Q 3’ 5’ 5’ 3’ l 5’ 3’ Q
Taq R

23. TaqMan Advantages Disadvantages Target specific fluorescence
Multiplexing
Disadvantages
High initial cost
Assay design not trivial
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.

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?

25. CFX 96 Real-Time PCR Detection System
• Modular thermal cycler platform, includes C1000 thermal cycler chassis, CFX96 optical reaction module, CFX Manager software .

26. 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 rate
5oC/sec
Average ramp rate
3.3oC/sec
Temp Accuracy
± 0.2oC
Temp Uniformity
± 0.4oC in 10 sec
Temp Range
0-100oC
The 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 ramping
10 second settling - the time it takes all wells to reach temperature - is unsurpassed in the market

27. Fast block architecture
Patented Block Design
Fast block architecture
To 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 Temperature
1000-Series Thermal Cycler Time to Temperature
You 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 Location
Uniform ramping + shorter settling times = Faster PCR

29. 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. 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. 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
Worry free performance over the lifetime of the instrument, LEDs do not burn out and they exhibit limited light degradation
No sacrificing a channel to normalize for positional bias with ROX, fluorescence is measured at the same distance for each well
Data 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 Channels
1-5 12
SYBR/FAM Only 1 3
FRET 6

33. Excellent Uniformity at 10l
Fast Scan
All Channels
Ave Ct = ± 0.12
Ave Ct = ± 0.12
A full 96 well plate was used for these experiments.

34. Flexibility to use 6 Filter Sets
Channel
Excitation (nm)
Detection (nm)
Calibrated Fluorophores 1
FAM™, SYBR Green I™ 2
VIC®, HEX™, TET™, Cal Gold 540™ 3
ROX™, TEXAS RED®, Cal Red 610™ 4
CY5, Quasar 670™ 5
Quasar 705™ 6
Accommodates FRET Chemistry
No need to recalibrate, ever. Reliable. Stable. Long life. Hassle free.

35. Unsurpassed Dye Separation
Achieve sensitive multiplexing by maximal excitation and detection of dyes 1 2 3 4 5
Fam
Hex
TxRed
Cy5
Q705
10000
20000
30000
40000
50000
60000
Channel
Signal
Fluorophore
Discrete Excitation and Detection of Dyes Even without color separating fluorescence between channels, there is almost no dye cross talk between channels
Using 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 10l
Ave Ct = ± 0.11
Max-Min =0.52
Ave Ct = ± 0.11
Max-Min =0.61
Hex
Texas Red
Again, excellent uniformity in all wells and all dye channels.

37. Excellent Uniformity at 10l in all channels
Ave Ct = ± 0.12
Max-Min =0.62
Ave Ct = ± 0.07
Max-Min =0.37
Cy5
Quasar 705
It 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 Guidelines
Thermal Gradient
Fast RT-PCR
Data Analysis

39. Melting temperature (Tm) of dsDNA
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
When 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 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. Melt Curve Analysis
Endpoint analysis to determine the melting temperature (Tm) of PCR products.

42. Melt Curve Analysis: Primer Dimer

43. Thermal Gradient
Used 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 concentration
Use temperature gradient feature
Look for lowest Ct value
Temperature
gradient
Dilution series of primer [ ]
SYBR Green I chemistry

45. Optimization of Annealing Temperature for Best Results
Annealing temperature is critical for
Specificity
Reproducibility
PCR Reaction Efficiency
Sensitivity
Reliable data
67oC
62oC
56oC
Efficiency = 68%
Efficiency = 99%
Efficiency = 98%
Serial dilutions
8 temps from 55oC to 68oC
62oC is optimal
-low Cts and highest reaction
efficiency
The 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 assay
Dynamic 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
Denaturation
Annealing
Extension

47. Quality Assays – SsoFast Eva Green Supermix

48. 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. SsoFast Eva Green Supermix: EvaGreen Dye
EvaGreen dye is similar to SYBR® Green I
Very low PCR inhibition
Increased sensitivity
Fast qPCR

50. Data Analysis:
Basic delta Ct
Delta-delta Ct
Pfaffl delta-delta Ct

51. (no normalization to reference gene)
Calculating for relative quantitation
Basic delta Ct method:
(no normalization to reference gene)
Primer set #2
Tissue #1: 22
Tissue #2: 24
Delta Ct:
24-22 = 2
Fold induction =
22 = 4

52. (assumes same efficiencies for each primer set)
Calculating for relative quantitation
Delta-delta Ct method:
(assumes same efficiencies for each primer set)
Reference Primer set
GOI Primer set
Tissue #1: 21 22
Tissue #2: 20 24
Delta Ct:
22-21 = 1
1st Delta
Delta Ct:
24-20 = 4
2nd Delta
Delta Ct:
4-1 = 3
Fold induction =
23 = 8

53. Ct SQ Calculating for relative quantitation
Problems of delta-delta Ct method: Ct 24 22
90% SQ

54. Ct SQ Calculating for relative quantitation
Problems of delta-delta Ct method: Ct 24 22
90%
100% SQ

55. Calculating for relative quantitation
Problem with the CT
Slopes are not parallel Ct 24 22
90%
100%
Starting quantity

56. Calculating for relative quantitation
Pfaffl method:
(Pfaffl, 2001; Nucleic Acid Research)
Pfaffl method:
(Pfaffl, 2001; Nucleic Acid Research)
Efficiencytarget deltaCt target (control-sample)
Efficiencytarget deltaCt target (control-sample)
Fold induction =
Fold induction =
Efficiencyreference deltaCt reference (control-sample)
Efficiencyreference deltaCt reference (control-sample)
Efficiency = 10-1/slope

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

58. Comparison of methods for relative quantitation calculations
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

59. Relative Gene Expression Analysis
What to Use as Standards
Plasmid DNA
PCR Product
Spiked sample (with plasmid or PCR product)
Positive cDNA control but unknown concentration (dilution)

60. There are no true “House keeping” genes
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:
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 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
Rethink PCR

62. CFX96 Real-Time PCR System
Questions?
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