2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 103

3. Add the images by selecting Menu item → “File” → “Add DIA
Batch item.”
4. Select the images which are to be analyzed.
5. Enter 10,000 as the number of estimated spots.
6. Check the “Include in BVA batch list” box.
7. Set the “Spot exclusion fi lter” to <30,000 for the volume.
8. Click “OK” to proceed.
9. Set up the experimental groups in the “BVA item settings”
window.
10. Select the most representative gel as master gel for matching.
11. Save the batch to the corresponding project.
12. Process the batch.

The BVA module allows some basic statistical analyses including:

3.6.5. Statistical Analysis

1. The determination of average intensity ratios between two
groups and the calculation of Student's t test p values.
2. The statistical analysis between all groups using One-Way
ANOVA (ANalysis Of VAriance).
3. Calculation of Two-Way ANOVA p values (statistical analysis
between the two conditions in an experiment with two inde-
pendent factors, e.g., time-dose studies).
4. The application of a false discovery rate (FDR) correction.
Perform the statistical analysis with the BVA module as follows:

1. Make sure the gel images are in the correct experimental
groups.
2. Switch to Protein mode.
3. Defi ne the statistics which should be calculated (Menu item
“Process” → “Protein statistics”). For comparisons of two
groups, choose “Average Ratio” and “Student's t test” and
select the groups which are to be compared in the “Population
1” and “Population 2” text box.
4. If more than two groups are compared, select additionally the
One-Way ANOVA. Should a Two-Way-ANOVA be required,
please refer to the DeCyder software manual.
5. If FDR correction is required, check the corresponding box
(see Note 14).

To select protein spots of interest in most cases, fi ltering must be
performed. If the Student's t test was applied, fi ltering of (1) aver-
age spot ratios between two groups and (2) p values should be
applied. When more than two groups are analyzed, fi ltering of
ANOVA p values is appropriate. Commonly, the fi lter is set to

3.6.6. Protein Filtering