2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 101

as well as the concept of IPS. In this section, image analysis with
the frequently used DeCyder 2D V7.0 software package (GE
Healthcare, Uppsala, Sweden) is described. The focus of this sec-
tion is not to replace the software manual but to briefl y outline the
principle procedure.

1. Take into account that DeCyder uses an Oracle database
(Oracle, Redwood Shores, CA, USA) installed on a dedicated
server to store images and all kinds of related data. Therefore,
be sure that you have a valid DeCyder account and appropriate
privileges to create your own projects and workspaces.
2. Login into DeCyder and generate a new project using the
“Organizer” tool to which images and data should be linked to.
3. If you want the images to be automatically grouped and named,
the DeCyder gel name recommendations should be followed.
The fi lename has to consist of a gel number (e.g., Gel 01), a
description of the function of the image (key terms: “Standard,”
“Control,” “Treated”), and the dye used. Instead of the key
terms, other words can be used but must be in brackets. For
example, the following two image fi les produced by the
Typhoon scanner will be grouped into a Gel named Gel 01:
File 1: Gel 01 Standard Cy3.gel.
File 2: Gel 01 (Sample name) Cy5.gel.
4. Start the Image Loader and select the project into which the
gel images are to be imported.
5. Add gel fi les to the import list.
6. If the fi les do not match the recommended names, perform
manual grouping as described in the manual.
7. Crop the images using the DeCyder image editor tool (or
ImageQuant, before importing the fi les into DeCyder) to
remove areas which contain information of no interest.
8. Ensure that only relevant spots remain inside the image and
that the patterns of the different images are similar.
9. Double-check settings (dye chemistry, assignment of fl uoro-
phores) and change these settings if required.
10. Import the selected image fi les.

3.6.1. Image Loading

The differential in-gel analysis (DIA) module processes images
from individual gels and performs (1) spot co-detection, (2) spot
quantifi cation and (3) normalization, and (4) in-gel intensity ratio
calculation of the spots from the Cy5 and Cy3 samples.
To perform a DIA analysis:

3.6.2. Differential In-Gel
Analysis

1. Open the DIA module within the DeCyder software.
2. Create a new DIA workspace and select all images of one gel.