Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 87)

Section 6 Data Analysis Module
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• Define Controls: Lists any positive or negative controls defined in the original plate
setup file. You may also add new controls or edit existing controls in this column by
clicking on the drop-down menu on the right side. The options are (+) Positive, (–)
Negative, or blank. Alternatively, you may type the letter ‘p’ or the plus sign (+) to select
a positive control, and you may also type the letter "n" or the minus sign (–) to select a
negative control. The controls specified in this column are used in the end point analysis
calculations that assign positive or negative values to the unknowns. You may click on
the Define Controls column header to sort the column by the controls
• Unknowns Call: If a well is not defined as a positive control or a negative control in the
Define Controls column, it is considered an unknown for end point analysis. This column
displays the value assigned to each unknown after Recalculate has been clicked.
Unknowns Call may be (+) Positive, (–) Negative, or blank. You may click on the
Unknowns Call column header to sort the table by Unknowns Call
• Unknowns Ranking: Lists the rank into which each unknown falls. An unknowns
ranking depends on the total number of ranks and the end RFUs value. You may click on
the Unknowns Ranking column header to sort the table by Unknowns Ranking
• Identifier: Lists the identifier for every well, as defined in the plate setup. You may click
on the Identifier column header to sort the table by identifier
6.8.3 Recalculate Button
Clicking Recalculate performs the final calculations used to determine unknown sample calls.
The Recalculate button is active when sufficient controls have been specified for the selected
method and End Point Tolerance settings. The following conditions result in an error message:
•
If the Positives method is selected and there is not at least one positive control defined in
the End Point analysis spreadsheet
•
If the Negatives method is selected and there is not at least one negative control defined
in the end point analysis spreadsheet
•
If the Positives & Negatives method is selected and there is not at least one positive
control and at least one negative control defined in the End Point Analysis spreadsheet
•
If no Controls are defined
• If the range is less than one (that is, the negative control RFU average is larger than the
positive control RFU average)
End point analysis cannot be performed if any of these error conditions is present when you click
Recalculate.
Once you are satisfied with your end point analysis, you may choose to view, save, or print a
customized End Point Analysis report. Click the Reports menu to obtain customized reports for
the end point data. Click Print to print the End Point analysis spreadsheet.
6.9 Gene Expression Analysis
The Gene Expression (Gene Expr) tab is used to evaluate relative differences in any target
concentration. For example, you can evaluate relative numbers of viral genomes or stably
transfected sequences. The most common application is evaluating target concentration in cDNA
samples to infer steady state messenger RNA levels.