BIO RAD CFX96 User Manual

Gene Expression Analysis
116
The efficiency (E) in the efficiency formulas refers to the “efficiencies” as described by Pfaffl
(2001) and Vandesompele et al. (2002). In these publications, an efficiency of 2 (perfect
doubling with every cycle) is equivalent to 100% efficiency in this software. You have the
option to convert your efficiency calculations to those used in the software by using the
following mathematical relationships:
• E = (% Efficiency * 0.01) + 1
• % Efficiency = (E - 1) * 100
Relative Quantity
The relative quantity (ΔC(t)) for any sample (GOI) is calculated with this formula:
Where:
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
•C
T (MIN)
= Average C(t) for the Sample with the lowest average C(t) for GOI
•C
T (sample)
= Average C(t) for the Sample
• GOI = Gene of interest (one target)
Relative Quantity When a Control Is Selected
When a control sample (control) is assigned, then the relative quantity (RQ) for any sample
(GOI) with a gene of interest is calculated with this formula:
Where:
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
•C
T (control)
= Average C(t) for the control sample
•C
T (sample)
= Average C(t) for any samples with a GOI
• GOI = Gene of interest (one target)
Standard Deviation of Relative Quantity
The standard deviation of the relative quantity is calculated with the following formula:
Where:
• SD Relative Quantity = standard deviation of the relative quantity
• SD C(t) sample = Standard deviation of the C(t) for the sample (GOI)
• Relative Quantity = Relative quantity of the sample
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
• GOI = Gene of interest (one target)
Relative Quantity
sample (GOI)
E
GOI
(C
T (MIN)
C–
T (sample)
)
=
Relative Quantity
sample (GOI)
E
GOI
C
T (control)
C–
T (sample)
()
=
SD Relative Quantity SD C(t)
GOI
Relative Quantity
Sample X
Ln (E
GOI
××)=

BIO RAD CFX96 Specifications

General

Temperature Uniformity	±0.4 °C
Software	CFX Maestro Software
Detection Method	Real-time PCR (qPCR)
Sample Capacity	96-well plate
Excitation Source	LED
Ramp Rate	Up to 5°C/sec
Heating Rate	5°C/sec
Sensitivity	Single copy detection
Power Requirements	100-240 V, 50/60 Hz
Temperature Range	0–100 °C