Real Time PCR Introduction and applications

1. 1
Perfect Results
a matter of knowledge
Mark N. Møller
Molecular Biologist and developer of RealQ Plus
Master Mix
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

2. 2
Ampliqon Real-Time Mixes
December 7, 2015 Mark N. Møller 2
RealQ Plus Green 2x Master Mix
• A323402 - Without ROX
• A324402 - Low ROX
• A325402 - High ROX
RealQ Plus Probe 2x Master Mix
• A313402 - Without ROX
• A314402 - Low ROX
• A315402 - High ROX
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

3. 3
Workflow
Sample
RNA isolation/
purification
DNA isolation/
purification
cDNA
synthesis
qPCR
Data
analysis
December 7, 2015 Mark N. Møller 3
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

4. 4
Disposition
Definitions and Concepts
Real-time PCR chemistry
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 4
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

5. 5
Definitions and Concepts
qPCR = Quantitative real-time PCR
Quantification not quantitation
Cq = Quantification cycle (Ct, Cp and TOP)
Reference genes = Genes used for normalization
December 7, 2015 Mark N. Møller 5
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

6. 6
Definitions and Concepts
Analytical Sensitivity:
”Minimum number of copies in a sample that
can be measured accurately with an assay”.
December 7, 2015 Mark N. Møller 6
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

7. 7
Definitions and Concepts
Analytical Specificity:
”the qPCR assay detecting the appropriate
target sequence rather than other, nonspecific
targets also present in a sample”.
December 7, 2015 Mark N. Møller 7
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

8. 8
Definitions and Concepts
Repeatability:
”the precision and robustness of the assay with the
same samples repeatedly analyzed in the same
assay. It may be expressed as the SD for the Cq
variance”
December 7, 2015 Mark N. Møller 8
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

9. 9
Definitions and Concepts
December 7, 2015 Mark N. Møller 9
Reproducibility:
”the variation in results between runs or between
different laboratories and is typically expressed as
the SD or CV of copy number or concentrations”
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

10. 10
Disposition
Definitions and Concepts
Real-time PCR chemistry
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Examples
Conclusions
December 7, 2015 Mark N. Møller 10
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

11. 11
Real-time PCR Chemistry
December 7, 2015 Mark N. Møller 11
RealQ Plus Green 2x Master Mix
• A323402 - Without ROX
• A324402 - Low ROX
• A325402 - High ROX
RealQ Plus Probe 2x Master Mix
• A313402 - Without ROX
• A314402 - Low ROX
• A315402 - High ROX
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

12. 12
Real-time PCR Chemistry
SYBR Green
• ≥ 1000-fold increase
• Fluorescent signal is proportional to the amount of dsDNA
• Binds all dsDNA (unspecific)
”the qPCR assay detecting the appropriate target sequence rather than other, nonspecific
targets also present in a sample”. - MIQE
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

13. 13
Real-time PCR Chemistry
Hydrolysis Probe
• TaqMan is a hydrolysis probe
• One reporter signal  One amplified target
• Fluorescence when intended target is amplified (Specific)
”the qPCR assay detecting the appropriate target sequence rather than other, nonspecific
targets also present in a sample”. - MIQE
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

14. 14
Real-time PCR Chemistry
ROX Passive Reference Dye
December 7, 2015 Mark N. Møller 14
Intended to normalize for non-PCR related fluorescence variations
ROX can correct for:
- Air bubbles
- Fluorescence variations because of concentration and volumes (limited)
- Fluorescence variations beacuse of sample effects (limited)
ROX can identify:
- Evaporation
- Poor mixing
- Some pipetting errors
Excitation spectrum Emission spectrum
585 nm 605 nm
IRAN Distributor , Viragene Akam Co .
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15. 15
15
Real-time PCR Chemistry
ROX Passive Reference Dye IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

16. 16
Disposition
Definitions and Concepts
Real-time PCR chemistry principles
Optimizing and analyzing data
• Primer optimization and PCR protocol
• Amplification Plot
• Standard Curves
• Multi Component Plot
• Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 16
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

17. 17
Optimizing and Analyzing Data
Primer optimization and PCR protocol
Primer design affects specificity, efficiency and accuracy.
• Find target sequence (NCBI)
• Upload correct sequence to primer software
• Primer 3
• http://primers.gene-quantification.info/
• PrimerExpress (ABI)
• Important parameters
• Melting temperature (TM)
• GC-content
• Target length (50-150 bp)
• Self priming
• Primer concentration (100-500 nM)
December 7, 2015 Mark N. Møller 17
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

18. 18
Optimizing and Analyzing Data
Primer optimization and PCR protocol
December 7, 2015 Mark N. Møller 18
Initial heating
15 min.
Cycling
15 -
30 sec.
30 sec.
30 sec.
Melt Curve
15 sec.
15 sec.
1 min.
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19. 19
40 °C 50 °C45 °C 55 °C 60 °C
Mismatch vs. perfect match
”Although primer optimization has become unfashionable, it is clear that poor annealing
optimization has a large effect on assay quality” - MIQE
Optimizing and Analyzing Data
Primer optimization and PCR protocol
A TT C G AA
T C
A T A AT C G A C AATT G A C G AATT
A A
A T C GT AA
T T T TC C CG G G GT TA AT T T TC C CA A A AT T T TC CA AG G
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

20. 20
Disposition
Definitions and Concepts
Real-time PCR chemistry
Optimizing and analyzing data
o Primer concentration and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 20
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

21. 21
Optimizing and Analyzing Data
The amplification plot
December 7, 2015 Mark N. Møller 21
0
0,5
1
1,5
2
2,5
3
3,5
4
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ΔRn
Cycle
Lag Phase
Plateau Phase
Linear Phase
Log Phase
Linear View vs. Log View
0,01
0,1
1
10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ΔRn
Cycle
Lag Phase
Log Phase
Linear Phase
Plateau Phase
Log phase is the most important phase!
IRAN Distributor , Viragene Akam Co .
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22. 22
Optimizing and Analyzing Data
Lag Phase - Baseline
December 7, 2015 Mark N. Møller 22
Rn dRn
• Spread lag phase
• Different cross points
• Flat lag phase
• One y-axis cross point in 0
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23. 23
Optimizing and Analyzing Data
Lag Phase - Baseline
December 7, 2015 Mark N. Møller 23
Too Low!
Increase the end cycle
Too high!
Decrease the end cycle and start
cycle
IRAN Distributor , Viragene Akam Co .
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24. 24
24
Optimizing and Analyzing Data
Lag Phase - Baseline
Baseline set correct!
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

25. 25
Optimizing and Analyzing Data
The amplification plot
December 7, 2015 Mark N. Møller 25
0
0,5
1
1,5
2
2,5
3
3,5
4
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ΔRn
Cycle
Lag Phase
Plateau Phase
Linear Phase
Log Phase
(Exponential)
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
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26. 26
Optimizing and Analyzing Data
Linear and Plateau Phase
December 7, 2015 Mark N. Møller 26
”After 30 cycles of PCR, well over 90 % of
primers and nucleotides are not consumed
and once the plateau is reached,
amplification cannot be restarted by adding
fresh reagents”
J. Hedman, doctoral thesis: ”DNA Analysis of PCR inhibitory forensic samples”,
2011.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

27. 27
27
Optimizing and Analyzing Data
Linear and Plateau Phase
Two main factors on linear and plateau phase:
• Target product inhibition of Polymerase
• Annealing competion: dsDNA self-annealing vs.
primer annealing
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
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28. 28
Optimizing and Analyzing Data
The amplification plot
December 7, 2015 Mark N. Møller 28
How can we use the amplification plot to optimize,
analyze and help us generate the perfect results?
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

29. 29
Optimizing and Analyzing Data
Log Phase – Defining Cq
December 7, 2015 Mark N. Møller 29
Second derivative maximum method:
Cq - value
Second derivative of
amplification plot
Amplification Plot
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

30. 30
Optimizing and Analyzing Data
Log Phase – Defining Cq
December 7, 2015 Mark N. Møller 30
0
0,5
1
1,5
2
2,5
3
3,5
4
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ΔRn
Cycle
Perfect
Fluorescence threshold – Linear View
Cq - value
Too high
Too Low
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

31. 31
0,01
0,1
1
10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ΔRn
Cycle
Optimizing and Analyzing Data
Log Phase – Defining Cq
December 7, 2015 Mark N. Møller 31
Perfect
Fluorescence threshold – Log View
Cq - value
Too high
Too Low
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32. 32
Let’s look at it
December 7, 2015 Mark N. Møller 32
Steep
Flat
(Inhibitors)
Modified from J. Hedman, doctoral thesis: ”DNA Analysis of PCR inhibitory forensic samples”, 2011.
IRAN Distributor , Viragene Akam Co .
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33. 33
Check list for perfect results
Amplification Plot
December 7, 2015 Mark N. Møller 33
 Sigmoidal curves with four phases
 Steep sigmoidal curve
 Standard deviation ≤ 0.2
between replicates
 Negative controls (NTC) within limits
(≥ 40 cycles)
 Consistent spacing (Standard curves)
Checklist:
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Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

34. 34
Break
34
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

35. 35
Disposition
Definitions and Concepts
Real-time PCR chemistry principles
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 35
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

36. 36
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 36
18
20
22
24
26
28
30
32
34
-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5
Cq
Log ng DNA
Standard Curve
0
1
2
3
4
5
6
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
ΔRn
Cycle
Amplification Plot
Efficiency = How efficient the polymerase amplifies the target.
1 cycle with 1 DNA doubling = 100 % efficiency
IRAN Distributor , Viragene Akam Co .
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37. 37
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 37
”Amplification efficiency should be determined
from the slope of the log-linear portion of the
calibration curve… The dynamic range should
cover at least 3 orders of magnitude and ideally
should extend to 5 or 6 log10 concentrations.”
S. Bustin et al.: ”The MIQE guidelines: Minimum Information for Publication of
Quantitative Real-Time PCR Experiments”, 2009.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

38. 38
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 38
PCR efficiency = 10−1/𝑠𝑙𝑜𝑝𝑒
− 1
18
20
22
24
26
28
30
32
34
-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5
Cq
Log ng DNA
Standard Curve
R2 = Statistical measure of how close the
data are fitted to the regression line (≥ 0.98)
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39. 39
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 39
For a precise standard curve, it important to:
- Prepare the dilution series from the previous tube (change filter tips in
between)
- Dip the tip as little as possible and pipet carefully and use low retention
tips
- Calibrate pipets and use sufficient volumes
- Have at least three replicates
- Mix thoroughly, but do not vortex nor pipet up and down when mixing
doublestranded DNA
- Use same DNA as target
IRAN Distributor , Viragene Akam Co .
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40. 40
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 40
How can we use the standard curve to optimize, analyze
and help us generate the perfect results?
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

41. 41
Optimizing and Analyzing Data
Standard curve
December 7, 2015 Mark N. Møller 41
Too low Efficiency Too high efficiency
PCR efficiency = 10−1/𝑠𝑙𝑜𝑝𝑒
− 1
18
20
22
24
26
28
30
0 0,5 1 1,5 2 2,5 3
Cq
Log ng DNA
y = -3.7411x + 29.142
R2 = 0.9781
18
20
22
24
26
28
30
0 0,5 1 1,5 2 2,5 3
Cq
Log ng DNA
y = -3.0767x + 28.277
R2 = 0.964
= 10−1/−3.7411 − 1
= 0.851
PCR efficiency = 10−1/𝑠𝑙𝑜𝑝𝑒
− 1
= 10−1/−3.0767
− 1
= 1.114
= 111.4%= 85.1 %
90 – 110 %
(-3.6 > Efficiency > -3.1)
100 % = -3.322
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42. 42
• Too low efficiency = reached sensitivity limit
• Too low efficiency = lack of annealing temperature or primer
concentration optimization
• Too low efficiency = lack of primer sequence optimization
• Too high efficiency = Inhibitors and lack of sample
concentration optimization
• Too high efficiency = Too high primer concentration
• Too high efficiency = Unspecific amplification
42
Optimizing and Analyzing Data
Standard curve
Sensitivity: ”Minimum number of copies in a sample that can be measured
accurately with an assay”.
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

43. 43
43
Optimizing and Analyzing Data
Standard curve
”PCR efficiency is particularly important when reporting mRNA
concentrations for target genes relative to those for reference genes.
The ΔΔCq method is one of the most popular means of determining
differences in concentrations between samples and is based on
normalization with a single reference gene…The 2 genes must be
amplified with comparable efficiencies for this comparison to be
accurate.” - MIQE
Relative quantification
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44. 44
Check list for perfect results
Standard Curve
December 7, 2015 Mark N. Møller 44
 Efficiency between 90-110 %
 R2 value ≥ 0.98
 Efficiency is not falsely high
 No banana shaped standard curve
Checklist:
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

45. 45
Disposition
Definitions and Concepts
Real-time PCR chemistry principles
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 45
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

46. 46
Optimizing and Analyzing Data
Multi component view
December 7, 2015 Mark N. Møller 46
ROX
SYBR
Amplification
Plot
Melt
Curve
Fluorescence raw data
IRAN Distributor , Viragene Akam Co .
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47. 47
Optimizing and Analyzing Data
Multi Component View
December 7, 2015 Mark N. Møller 47
How can we use the multi component view to optimize,
analyze and help us generate the perfect results?
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

48. 48
Optimizing and Analyzing Data
Multi component view
December 7, 2015 Mark N. Møller 48
Low SYBR signal:
• Failing bulb
• Mix expiration
High SYBR signal
• Can inhibit PCR
ROX signal inconsistency:
• Evaporation
• Poor mixing
• Some pipetting errors
Cq Values:
• Manipulative with SYBR
• Manipulative with ROX
ROX
SYBR
Cq difference
IRAN Distributor , Viragene Akam Co .
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49. 49
Check list for perfect results
Multi Component View
December 7, 2015 Mark N. Møller 49
 Reporter within range and at the desired
levels
 ROX passive reference dye signal
is unchanged
 ROX level correct and help maintain
low standard deviation
Checklist:
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

50. 50
Disposition
Definitions and Concepts
Real-time PCR chemistry principles
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 50
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

51. 51
Optimizing and Analyzing Data
Melt Curve
December 7, 2015 51
Intensity
Temperature
TM
50 %
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52. 52
52
50 °C 95 °C
FluorescenceIntensity
Optimizing and Analyzing Data
Melt Curve
Temperature
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53. 53
53
dI/dT
Temperature
TM
Mark N. Møller
Temperature
- dI/dT
TM
Optimizing and Analyzing Data
Melt Curve
Intensity
Temperature
TM
50 %
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54. 54
Optimizing and Analyzing Data
Melt Curve
December 7, 2015 Mark N. Møller 54
Target
Melt point influenced by:
• DNA amount
• pH
• Buffer composition
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55. 55
55
Optimizing and Analyzing Data
Melt Curve
Different buffers can give different TM
=> Optimize annealing temperature to your
specific buffer
IRAN Distributor , Viragene Akam Co .
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56. 56
Optimizing and Analyzing Data
Melt Curve
December 7, 2015 Mark N. Møller 56
How can we use the melt curve to optimize, analyze and
help us generate the perfect results?
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

57. 57
Optimizing and Analyzing Data
Melt Curve
December 7, 2015 Mark N. Møller 57
Unspecific
product
Target
• Redesign primers
• Optimize annealing temperature
• Optimize primer concentration
Target
• Have separate work spaces
• Setup
• PCR run
• Opening plates
+ NTC
(contamination)
Unspecific
product
IRAN Distributor , Viragene Akam Co .
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58. 58
Optimizing and Analyzing Data
Melt Curve
December 7, 2015 Mark N. Møller
58
Primer
dimers
Target
• Redesign primers
• Optimize annealing temperature
• Hot Start enzyme
Primer dimer formation
IRAN Distributor , Viragene Akam Co .
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59. 59
Check list for perfect results
Melt Curve
December 7, 2015 Mark N. Møller 59
 Clear and distinguished peak at intended
temperature
 No primer dimers
 Negative NTC’s
 No unspecific amplification
Checklist:
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Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

60. 60
Break
60
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

61. 61
Disposition
Definitions and Concepts
Real-time PCR chemistry principles
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 61
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

62. 62
Example
Absolute Quantification
December 7, 2015 Mark N. Møller 62
0
1
2
3
4
5
6
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
ΔRn
Cycle
Amplification Plot
SD = 0.07
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

63. 63
Example
Absolute Quantification
December 7, 2015 Mark N. Møller 63
18
20
22
24
26
28
30
32
34
-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5
Ct
Log ng DNA
Standard Curve
Slope: -3.376
R2: 0.999
PCR efficiency = 10−1/𝑠𝑙𝑜𝑝𝑒 − 1
= 10−1/−3.376 − 1
= 0.978
= 97.8 %
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

64. 64
Example
Absolute Quantification
December 7, 2015 Mark N. Møller 64
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

65. 65
Example
Absolute Quantification
December 7, 2015 Mark N. Møller 65
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
60 65 70 75 80 85 90 95 100
DerivativeReporter(-R')
Temperature (°C)
Melt Curve
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

66. 66
Disposition
Definitions and Concepts
Real-time PCR chemistry
Optimizing and analyzing data
o Primer optimization and PCR protocol
o Amplification Plot
o Standard Curves
o Multi Component Plot
o Melt Curve
Example
Conclusions
December 7, 2015 Mark N. Møller 66
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

67. 67
Conclusions
To get the best results it is important to know:
• How to set up and optimize your experiment
• How to analyze your results
• This can be done with Ampliqon RealQ Plus
Master Mixes!
December 7, 2015 Mark N. Møller 67
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .

68. 68
December 7, 2015 Mark N. Møller 68
IRAN Distributor , Viragene Akam Co .
Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .