BIO RAD CFX384, Instruction Manual

Page: 127 / 158

Gene Expression Analysis
118
Standard Deviation for the Normalized Expression
Re-scaling the normalized expression value is accomplished by dividing the standard
deviation of the normalized expression by the normalized expression value for the highest or
lowest individual expression levels, depending on the Scaling Option you choose. The
standard deviation (SD) of the normalization factor is calculated with this formula:
Where:
• RQ = Relative quantity of a sample
• SD = Standard deviation
• NF = Normalization factor
• Ref = Reference target
• n = Number of reference targets
When a control sample is assigned, you do not need to perform this re-scaling function on the
standard deviation, as shown in the following formula:
Where:
• NE = Normalized Expression
• RQ = Relative quantity of a sample
• SD = standard deviation
• GOI = Gene of interest (one target)
Normalized Expression Scaled to Highest 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 highest level of expression
in all the samples. The software sets the highest level of expression to a value of 1, and re-
scales all the sample expression levels. The highest scaling is calculated by this formula:
Where:
• GOI = Gene of interest (target).
SD NF
n
NF
n
SD RQ
sample (Ref 1)
n RQ
sample (Ref 1)
×
---------------------------------------------
⎝ ⎠
⎛ ⎞
2
SD RQ
sample (Ref 2)
n RQ
sample (Ref 2)
×
---------------------------------------------
⎝ ⎠
⎛ ⎞
2
…
SD RQ
sample (Ref n)
n RQ
sample (Ref n)
×
---------------------------------------------
⎝ ⎠
⎛ ⎞
2
+ + +
×
=
SD NE
sample (GOI)
NE
sample (GOI)
SD NF
sample
NF
sample
------------------------------
⎝ ⎠
⎛ ⎞
2 SD RQ
sample (GOI)
RQ
sample (GOI)
-------------------------------------------
⎝ ⎠
⎛ ⎞
2
+
×
=
Scaled Normalized Expression
sample (GOI)
Normalized Expression
sample (GOI)
Normalized Expression
Highest sample (GOI)
--------------------------------------------------------------------------------------------------
=