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Patricia de Winter
University College London and qStandard
What is qPCR quantification?
• The method by which the amount of a DNA
template initially present can be measured
by monitoring qPCR amplification curves
100
90
80
70
Fluorescence
60
50
40
30
20
Threshold line
10
0
0 5 10 15 20 25 30 35 40
Cycle number
Cq determination - cycle threshold
13.67 15.51
20% of
curve
height Velocity, m/s
(1st derivative)
14.9 16.8
Acceleration, m/s2
(2nd derivative)
Cq
8
7
Amplicon
length 6
50 150 250 350 450 550
Gene 1 69 amplicon length (bp)
Gene 2 70
Measured Cq
Gene 3 73
11
Gene 4 91
10
Gene 5 112 9
Cq
Gene 6 117 8
7
Gene 7 244
6
Gene 8 266 50 150 250 350 450 550
amplicon length (bp)
Gene 9 468
Detection of fluorescence
by the hardware
7
10 mACTB
107 copies of standard
mmu Actb on RG and LC2
standard run
on RG6000 and LightCycler1.2
RG is
more sensitive
by ~2-3 cycles
Fluorescence
0 10 20 30 40
Cycle
Cycle threshold
E=100% lower
lowest
PCR efficiency
• Can be expressed in three ways:
In this example:
E = 10(-1/slope)
Efficiency =10(-1/-3.330)
=1.0 or 100%
Determination of PCR efficiency:
cDNA dilution series
Sample #1
25 y = -3.3577x + 29.913
R² = 1 • Four 5-fold dilutions of each cDNA
Cq
20
15
1.00 2.00 3.00 4.00 5.00
prepared
• Provides individual sample
log copy no. /rxn
Sample #2
25 y = -3.3682x + 29.938
R² = 1
efficiency – direct measure
Cq
20
15
1.0 2.0 3.0
log copy no./rxn
4.0 5.0
• Standard curve efficiency was 98%
Sample #3 Slope Efficiency, E = 10 (-1/slope) Efficiency (%)
25 y = -3.3695x + 29.948
R² = 1 -3.3577 1.9853 98.53
Cq
20
-3.3682 1.9810 98.10
15
1.0 2.0 3.0 4.0 5.0 -3.3695 1.9805 98.05
log copy no./rxn
-3.3509 1.9880 98.80
25
Sample #4
-3.3682 1.9810 98.10
y = -3.3692x + 29.946
R² = 1 -3.3637 1.9829 98.29
Cq
20
2.5
120
2
100 1.5
1
80
log Fluorescence
0.5
Fluorescence
0
60
0 5 10 15 20 25 30 35 40
-0.5
Efficiency = 10-1/slope
40
-1 2.04 (104%)
-1.5 1.94 (94%)
20
-2
1.96 (96%)
1.93 (93%)
0 -2.5
0 5 10 15 20 25 30 35 Cycle number
40 1.84 (84%)
Cycle number
Why worry about efficiency?
107
101
Before After
Mean 197,685 189,381
s 68,133 13,645
%CV 34.5 7.2
Relative quantification - DDCt
• The classic relative quantification model,
“delta-delta Ct” subtracts the Cq of a
sample from that of a calibrator, and 2 is
then raised to the power of this value:
2DCtGOI(calibrator-sample)
Normalised Relative Quantity =
2DCtrefgene (calibrator-sample)
Worked example
226.0-21.0 = 25 = 32
NRQ = = 2.82
220.0-16.5 = 23.5 = 11.31
GOI
Ref gene
Problem
Relative quantification (Pfaffl method)
• Pfaffl (2001) modified the delta-delta Ct method
to include the assay efficiency for each gene. E
can be determined from a dilution series of pooled
cDNA or by an indirect method
(EGOI) DCT(GOI control-sample)
Normalised Relative Quantity =
(Erefgene) DCT(refgene control-sample)
Worked example
226.0-21.0 = 25 = 32
NRQ = = 3.44
1.8920.0-16.5 = 1.893.5 = 9.28
Workshops:
qPCR
Services:
Custom assay design and preparation of
qPCR standards