BIO RAD CFX384, Instruction Manual

Page: 128 / 158

CFX96 and CFX384 Systems Manual
119
Normalized Expression Scaled to Lowest Expression
Level
When the experiment does not include controls, scale the normalized expression (NE) for each
target (gene) by dividing the expression level of each sample by the lowest level of expression
in all the samples. The software sets the lowest level of expression to a value of 1, and re-
scales all the sample expression levels. The lowest scaling is calculated by this formula:
Where:
• GOI = Gene of interest (target).
Standard Deviation for the Scaled Normalized Expression
Re-scaling the scaled normalized expression (NE) value is accomplished by dividing the
standard deviation (SD) of the normalized expression by the normalized expression value for
the highest (MAX) or lowest (MIN) expression level, depending on which scaling option you
choose.
NOTE: When a control sample is assigned, you do not need to perform this
rescaling function on the standard deviation.
The formula for this calculation is shown here:
Where:
• NE = Normalized expression
• SD = Standard deviation
• GOI = Gene of interest (target)
• MAX = Highest expression level
• MIN = Lowest expression level
Corrected Values Formulas
A difference between corrected values and noncorrected values is only seen if a standard
curve is created as part of the real-time PCR experiment. The software uses three equations in
determining the error propagation:
• Standard Error
• Standard Error for Normalized Expression
• Standard Error for the Normalized Gene of Interest (target)
The formula for standard error is shown here:
Where
• n = Number of reference targets (genes)
• SD = Standard deviation
Scaled Normalized Expression
sample (GOI)
Normalized Expression
sample (GOI)
Normalized Expression
Lowest sample (GOI)
-------------------------------------------------------------------------------------------------
=
SD Scaled NE
sample (GOI)
SD NE
sample (GOI)
NE
MAX or MIN (GOI)
-----------------------------------------------
=
Standard Error
SD
n
-------
=