Bio-Rad Bio-Plex Software® Upgrades and Conversions - Manual (Page 235)

Normalization Formulas
223
Normalization Formulas
Bio-Plex Manager uses the following formulas to calculate normalization
values.
• All calculations are done first on a per well basis
• All fluorescence values are adjusted mean fluorescence
(Fl – Background), unless no blanks are provided in the experiment
• All calculations are done on a per gene level, except where
indicated
• The following equations are written for gene expression studies.
You can substitute “gene” with “analyte” and use the same
calculations for normalizing protein concentrations with internal/
endogenous controls
For these calculations:
Gene of Interest = Analyte of Interest
Housekeeping Gene = Endogenous Control Protein
Variables below are HKG = Housekeeping Gene
FI = Fluorescence Intensity
n = number of housekeeping genes
Average FI
(sample)
= FI
well1
, FI
well2
. . . FI
welln
Norm Ratio is the software’s term for normalized expression values. It is
calculated three different ways, depending on the options chosen in the
Normalization Settings dialog. These are outlined below as Norm Ratio, Norm
Ratio’ and Norm Ratio’’.
A control sample may be assigned in order to:
• Compare results across multiple plates (control sample must be
the same biological sample on each plate)
• Simply express all results on the same plate, relative to the controls
sample
BioPlex_6.book Page 223 Friday, September 23, 2011 11:55 AM