Bio-Rad Firmware & Software Updates - Manual (Page 113)

Gene Expression Analysis
102
For the software to recognize a sample as an inter-plate calibrator, it must share matching
target name, sample name, and if used, collection name, across every plate being compared.
NOTE: At least one inter-run calibrator sample must be present in the Gene Study
for inter-run calibration to occur. Targets without appropriate inter-run calibrator
samples will be processed without correction in the Gene Study (not
recommended).
During inter-run calibration an algorithm is used to calculate the pair-wise differences between
the C
q
values (C
q
) for all samples that qualify as inter-run calibrators. All data within the Gene
Study are normalized by inter-run calibrator to calculate the smallest average C
q
value. When
the data files within the Gene Study include more than one inter-run calibrator, then the
calibrator with the smallest average C
q
value becomes the dominant inter-run calibrator. The
dominant calibrator is used to adjust all C
q
values in the Gene Study.
To find the dominant inter-run calibrator, the software calculates the average of the C
q
values
for all inter-run calibrators of a given target, and then uses a multitiered algorithm to determine
the dominant inter-run calibrator within all the data. The algorithm for finding the dominant
inter-run calibrator includes the following hierarchy:
1. Set the dominant calibrator to the target with the highest number of common replicate
groups in a given pair-wise comparison.
2. If any target has the same number of common replicate groups, then set the dominant
calibrator to the target with the smallest range of C
q
values in pair-wise comparisons.
The range is examined by comparing the absolute value of the difference between the
maximum and minimum C
q
for the inter-run calibrators of a given target.
3. If any target has an identical range as the C
q
values, then set the dominant calibrator to
the target with the smallest absolute value of average C
q
for eligible inter-run calibrator
samples.
4. If any target has identical average C
q
absolute values, then set the dominant calibrator
to the replicate group with the smallest C
q
.
NOTE: The first data file imported into the Gene Study will always serve as the
“hub” file for pairwise data comparison during inter-run calibration.
Gene Study Window
The Gene Study window includes two tabs:
• Study Setup tab. Click this tab to manage the runs in the Gene Study. Adding or
removing data files in a Gene Study does not change the original data in that file
• Study Analysis tab. Click this tab to view the gene expression data for the combined
runs