CFX96 Real-Time PCR Detection System

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.

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
Log Target DNA
In theory, the amount of DNA produced at every
cycle should double, Product(T) (Template0) x
2n (n of cycles)
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
Log View
11
Mathematical Implications
Ideal PCR
ProductT(Template0)2n Where nNumber of Cycles

• 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

14
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
l
l
19
SYBR Green I

• Advantages
• Experiment only requires primers
• Disadvantages
• Potential contribution to fluorescence from
  non-specific products (primer-dimers)
• No multiplexing

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 TaqManTM
Add iQ Supermix, Hybridization Probe and sample
d.NTPs
Primers
Thermal Stable DNA Polymerase
Denaturation
l
Annealing
22
Cleavage-based assay TaqManTM
Extension Step
l
23
TaqMan

• Advantages
• Target specific fluorescence
• Multiplexing
• Disadvantages
• High initial cost
• Assay design not trivial

24
Real Time PCR Technology
Real-Time PCR -Enables detection and
quantification of sample -Extremely
sensitive -Can be used in various applications
(gene expression, allelic discrimination,
pathogen detection)
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
27
Patented Block Design
Fast block architecture
Mass-reduced sample block
Patented by Bio-Rad
28
Time to Temperature
1000-Series Thermal Cycler Time to Temperature
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
• gt100/well/scan
• Laser Homing of shuttle at every scan

32
Multiple Fast Scan Modes
Mode Channel(s) Scan Time (sec)
All Channels 1-5 12
SYBR/FAM Only 1 3
FRET 6 3
33
Excellent Uniformity at 10?l
34
Flexibility to use 6 Filter Sets
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.
35
Unsurpassed Dye Separation

• Achieve sensitive multiplexing by maximal
  excitation and detection of dyes

36
Excellent Uniformity at 10??l
37
Excellent Uniformity at 10?l in all channels
38
CFX96 features for Reaction Optimization

• Melt Curve MIQE Guidelines
• Thermal Gradient
• Fast RT-PCR
• Data Analysis

39
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
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

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

1. annealing temperature
2. primer concentration

• Use temperature gradient feature
• Look for lowest Ct value

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

• Serial dilutions
• 8 temps from 55oC to 68oC
• 62oC is optimal
• -low Cts and highest reaction
• efficiency

46
Fast PCR 3-step PCR vs 2-step PCR
Annealing
Extension
Denaturation
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 gt90C)
• 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
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
22 4
Fold induction
52
Calculating for relative quantitation
Delta-delta Ct method (assumes same
efficiencies for each primer set)
Reference Primer set
GOI Primer set
21
Tissue 1
22
Tissue 2
20
24
Delta Ct
22-21 1
1st Delta
Delta Ct
24-20 4
Delta Ct
4-1 3
2nd Delta
Fold induction
23 8
53
Calculating for relative quantitation
Problems of delta-delta Ct method
Ct
24
22
90
SQ
54
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
21
Tissue 1
22
Tissue 2
20
24
90 1.9
Efficiency
100 2
(From Standard curve)
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
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/genor
  m/
• 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-Rads 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
CFX96 Real-Time PCR System
Questions?
Thank you for joining us!