CFX96 Real-Time PCR
Detection System
Fast, Friendly, Flexible
Designed for the Way You Work
Rethink PCR
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Discussion for today
AMPLIFICATION
• Real time PCR technology
• CFX96
• CFX96 system features
– Methods for optimization
– Data Analysis
• CFX96 software
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What is Real-Time qPCR?
AMPLIFICATION
• 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.
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Limitations of standard PCR
AMPLIFICATION
Amplification is exponential, but the exponential increase
is limited:
A linear increase follows
exponential phase
Theoretical
•
Eventually plateaus
In theory, the amount of DNA
produced at every cycle should
double,
Product(T) = (Template0) x 2n
(n = # of cycles)
Log Target DNA
•
Real Life
Cycle #
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AMPLIFICATION
Standard PCR is as endpoint
96 identical reactions will have very different final
amounts of fluorescence at endpoint
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AMPLIFICATION
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.
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AMPLIFICATION
How is quantitative data collected?
Log Target DNA
Theoretical
Detector
Cycle #
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Real Life
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AMPLIFICATION
Threshold Cycle, CT
96 identical reactions will have
almost identical CT values
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AMPLIFICATION
Threshold Cycle, CT
The point at which the fluorescence rises
appreciably above background
Threshold can be placed anywhere in the exponential
(log-linear) phase
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Threshold Setting
AMPLIFICATION
• After baseline subtraction, a threshold line is set empirically or by a
statistical calculation at a fluorescence value above background.
Threshold
Log
View
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Mathematical Implications
AMPLIFICATION
Ideal PCR
n
ProductT=(Template0)2
Where n=Number of Cycles
•
1 CT Difference = 2 fold difference in starting template amount
•
3.3 CT Difference = 10 fold difference in starting template amount
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AMPLIFICATION
•
Threshold Cycle, CT
Correlates strongly with the starting copy number
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Threshold Cycle, CT
AMPLIFICATION
•
Correlates strongly with the starting copy number
2n = 10 fold
n ln 2 = ln 10
n = ln10
ln 2
n = 3.32
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Real-Time PCR: Applications
AMPLIFICATION
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)
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AMPLIFICATION
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From CT values, we can determine
the initial copy number
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AMPLIFICATION
Chemistries used in real time PCR
• Intercalation Dyes
• Hybridization Probes
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AMPLIFICATION
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
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Intercalation Dyes:
AMPLIFICATION
SYBR Green I
l
l
l
Taq
3’
ID
ID
ID
ID
Taq
l
l
Version 1.0
5’
ID
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3’
SYBR Green I
AMPLIFICATION
• Advantages
– Experiment only requires primers
• Disadvantages
– Potential contribution to fluorescence from nonspecific products (primer-dimers)
– No multiplexing
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Hybridization Probes
AMPLIFICATION
Currently, hybridization probe strategies
fall into three main categories:
• 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
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Cleavage-based assay: TaqManTM
AMPLIFICATION
5’
3’
5’
3’
Add iQ Supermix,
Hybridization Probe
and sample
d.NTPs
Primers
Thermal Stable
DNA Polymerase
R
5’
Probe
Q
3’
Denaturation
Taq
l
R
5’
Annealing
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Q
3’
Cleavage-based assay: TaqManTM
AMPLIFICATION
Q
R
5’
3’
R
Extension Step
Q
Taq
5’
3’
R
Q
Taq
5’
5’
3’
R
Taq
5’
5’
3’
l
R
Taq
5’
5’
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3’
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TaqMan
AMPLIFICATION
• Advantages
– Target specific fluorescence
– Multiplexing
• Disadvantages
– High initial cost
– Assay design not trivial
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Real Time PCR Technology:
AMPLIFICATION
Real-Time PCR:
-Enables detection and quantification of sample
-Extremely sensitive
-Can be used in various applications (gene expression,
allelic discrimination, pathogen detection)
Questions?
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AMPLIFICATION
CFX 96 Real-Time PCR
Detection System
• Modular thermal cycler
platform, includes
C1000 thermal cycler
chassis, CFX96 optical
reaction module, CFX
Manager software
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Unsurpassed Thermal Cycling
AMPLIFICATION
• 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
Version 1.0
0-100oC
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Patented Block Design
AMPLIFICATION
Fast block architecture
Mass-reduced sample block*
* Patented by Bio-Rad
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Time to Temperature
AMPLIFICATION
1000-Series Thermal Cycler Time to Temperature
Probe Location
Uniform ramping + shorter settling times = Faster PCR
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CFX96 Optical Technology
AMPLIFICATION
• 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
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Next Generation Optical
Technology:
AMPLIFICATION
• 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
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AMPLIFICATION
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
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Multiple Fast Scan Modes
AMPLIFICATION
Version 1.0
Mode
Channel(s)
Scan Time (sec)
All Channels
1-5
12
SYBR/FAM Only
1
3
FRET
6
3
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Excellent Uniformity at 10l
AMPLIFICATION
Fast Scan
All Channels
Ave Ct = 19.29 ± 0.12
Ave Ct = 19.81 ± 0.12
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Flexibility to use 6 Filter Sets
AMPLIFICATION
Channel
Excitation (nm)
Detection (nm)
Calibrated Fluorophores
1
450-490
515-530
FAM™, SYBR Green I™
2
515-535
560-580
VIC®, HEX™, TET™, Cal Gold 540™
3
560-590
610-650
ROX™, TEXAS RED®, Cal Red 610™
4
620-650
675-690
CY5, Quasar 670™
5
672-684
705-730
Quasar 705™
6
450-490
560-580
Accommodates FRET Chemistry
No need to recalibrate, ever. Reliable. Stable. Long life. Hassle free.
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Unsurpassed Dye Separation
AMPLIFICATION
Achieve sensitive multiplexing by maximal excitation and detection of dyes
60000
50000
40000
Signal
30000
20000
Q705
10000
Cy5
TxRed
0
Hex
1
2
Fam
3
4
Channel
Version 1.0
5
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Fluorophore
Excellent Uniformity at 10l
AMPLIFICATION
Version 1.0
Hex
Texas Red
Ave Ct = 19.67 ± 0.11
Max-Min =0.52
Ave Ct = 19.21 ± 0.11
Max-Min =0.61
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Excellent Uniformity at 10l in all
channels
AMPLIFICATION
Cy5
Quasar 705
Ave Ct = 19.96 ± 0.12
Max-Min =0.62
Version 1.0
Ave Ct = 19.27 ± 0.07
Max-Min =0.37
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CFX96 features for Reaction
Optimization
AMPLIFICATION
• Melt Curve –MIQE Guidelines
• Thermal Gradient
• Fast RT-PCR
• Data Analysis
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Melt Curve Analysis
AMPLIFICATION
• 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
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Melt Curve Analysis
AMPLIFICATION
• 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
Double
Stranded DNA
Single
Stranded
Tm
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Melt Curve Analysis
AMPLIFICATION
Endpoint analysis to determine the melting
temperature (Tm) of PCR products.
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AMPLIFICATION
Version 1.0
Melt Curve Analysis:
Primer Dimer
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Thermal Gradient
AMPLIFICATION
•
•
•
Version 1.0
Used for one-step reactiontemperature 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.
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Thermal Gradient
AMPLIFICATION
1) annealing temperature
• Use temperature gradient feature
2) primer concentration
• Look for lowest Ct value
Dilution series of primer [ ]
Temperature
gradient
Version 1.0
SYBR
Green I chemistry
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Optimization of Annealing
Temperature for Best Results
AMPLIFICATION
• Annealing temperature is critical for
Specificity
Reproducibility
PCR Reaction Efficiency
Sensitivity
Reliable data
67oC
Efficiency = 68%
62oC
Efficiency = 99%
56oC
Efficiency = 98%
• Serial dilutions
8 temps from 55oC to 68oC
• 62oC is optimal
-low Cts and highest reaction
efficiency
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Fast PCR: 3-step PCR vs
2-step PCR
AMPLIFICATION
95ºC
72ºC
58ºC
Denaturation
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Annealing
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Extension
AMPLIFICATION
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Quality Assays – SsoFast Eva
Green Supermix
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SsoFast Eva Green Supermix:
AMPLIFICATION
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
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SsoFast Eva Green Supermix:
EvaGreen Dye
AMPLIFICATION
•EvaGreen dye is similar to SYBR® Green I
•Very low PCR inhibition
•Increased sensitivity
•Fast qPCR
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Data Analysis:
AMPLIFICATION
•Basic delta Ct
•Delta-delta Ct
•Pfaffl delta-delta Ct
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Calculating for relative quantitation
AMPLIFICATION
Basic delta Ct method:
(no normalization to reference gene)
Primer set #2
22
24
Tissue #1:
Tissue #2:
Delta Ct:
Fold induction =
Version 1.0
24-22 = 2
22 = 4
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Calculating for relative quantitation
AMPLIFICATION
Delta-delta Ct method:
(assumes same efficiencies for each primer set)
Reference Primer set
Tissue #1:
21
Tissue #2:
1st
Delta
2nd Delta
20
24
Delta Ct:
22-21 = 1
Delta Ct:
24-20 = 4
Delta Ct:
4-1 = 3
Fold induction = 23 = 8
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GOI Primer set
22
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Calculating for relative quantitation
AMPLIFICATION
Problems of delta-delta Ct method:
Ct
24
22
90%
SQ
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Calculating for relative quantitation
AMPLIFICATION
Problems of delta-delta Ct method:
Ct
24
22
90%
100%
SQ
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Calculating for relative quantitation
AMPLIFICATION
Problem with the CT
Slopes are not parallel
Ct
24
22
90%
100%
Starting quantity
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Calculating for relative quantitation
AMPLIFICATION
Pfaffl method:
(Pfaffl, 2001; Nucleic Acid Research)
Efficiencytarget
deltaCt target (control-sample)
Fold induction =
Efficiencyreference
deltaCt reference (control-sample)
Efficiency = 10-1/slope
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Calculating for relative quantitation
AMPLIFICATION
Pfaffl method:
(efficiencies are normalized)
Primer set #1Reference
(From Standard curve)
Tissue #1:
21
22
Tissue #2:
20
24
Efficiency:
90% = 1.9
Delta Ct:
20-21 = -1
Fold induction =
2target
deltaCt target
1.9reference
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Primer set #2 GOI
(24-22 = 2)
deltaCt reference
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=
(20-21 = -1)
100% = 2
24-22 = 2
4
0.53
=
7.5
AMPLIFICATION
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
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Relative Gene Expression Analysis
AMPLIFICATION
What to Use as Standards
•Plasmid DNA
•PCR Product
•Spiked sample (with plasmid or PCR product)
•Positive cDNA control but unknown concentration
(dilution)
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Vandesompele Method
AMPLIFICATION
• 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/
– 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
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Bio-Rad: Experts in Real-time PCR
AMPLIFICATION
• 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
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Rethink PCR
CFX96 Real-Time PCR System
AMPLIFICATION
Questions?
Thank you for joining us!
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