BIO RAD CFX384, Instruction Manual

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CFX96 and CFX384 Systems Manual
117
Normalization Factor
The denominator of the normalized expression equation is referred to as the normalization
factor. The normalization factor is the geometric mean of the relative quantities of all the
reference targets (genes) for a given sample, as described in this formula:
Where:
• RQ = Relative quantity
• n = Number of reference targets
• GOI = Gene of interest (one target)
Normalized Expression
Normalized expression (
ΔΔ
C(t)) is the relative quantity of your target (gene) normalized to the
quantities of the reference targets (genes or sequences) in your biological system. To select
reference targets, open the Experiment Settings window and click the reference column for
each target that serves as a reference gene.
The calculation for normalized expression is described in the following formula, which uses the
calculated Relative Quantity (RQ) calculation:
Where:
• RQ = Relative Quantity of a sample
• Ref = Reference target in an experiment that includes one or more reference targets
in each sample
• GOI = Gene of interest (one target)
Provided that reference targets do not change their expression level in your biological system,
the calculation of normalized expression will account for loading differences or variations in
cell number that is represented in each of your samples.
Normalized Expression When a Control Is Selected
When you select a control sample in the Experiment Settings window, the software sets the
expression level of the control sample to 1. In this situation, the software normalizes the
relative quantities of all target (gene) expression to the control quantity (a value of 1). This
normalized expression is equivalent to the unscaled normalized expression analysis when a
control is chosen.
Normalization Factor
sample (GOI)
(RQ
sample (Ref 1)
RQ
sample (Ref 2)
…
RQ
sample (Ref n)
)
× × ×
1
n
---
=
Normalized Expression
sample (GOI)
RQ
sample (GOI)
RQ
sample (Ref 1)
RQ
sample (Ref 2)
…
RQ
sample (Ref n)
× × × ( )
1
n
---
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=