Molecular Devices SpectraMax Paradigm User Manual page 31

Analyzing Fluorescence Intensity Data
Fluorescence intensity data is dependent on a number of variables. Raw data is compared to
a standard curve with known concentrations of a reference label.
A standard curve consists of, at a minimum, a blank sample and a reference standard sample
of known concentration. The raw data can then be expressed in equivalent concentration of
a reference label.
Analyzing and validating fluorescence intensity data generally consists of the following:
Background Correction and Quantification on page 31
Detection Limit on page 32
Linearity and the Linear Dynamic Range on page 32
Background Correction and Quantification
A blank well contains everything used with the sample wells except the label and sample-
specific compounds. Do not use an empty well for a blank.
The blank sample reveals the offset underlying each data sample. This offset does not carry
information on the label, and is generally subtracted before data reduction is done.
The blank-subtracted raw data are proportional to the quantity of label in a sample such that
the label concentration is quantified by the following equation.
conc
where conc
average values of replicates for the sample, blank, and standard wells. In the general case
where the standard curve covers a concentration range of more than a few linear logs,
(standard – blank) / conc
concentration of the label is determined by (sample – blank) / (slope of standard curve).
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
5014038 E
(sample – blank)
=
label
std – blank
conc
std
is the concentration of the standard, and sample, blank, and standard are
std
is equivalent to the slope of the standard curve, and so the
std
Chapter 2: Read Modes and Read Types
31