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Organizing Gel Images

We start with the raw data files on the hard disk. The example experiment consists of a control sample, and two samples 1 and 10 minutes after treatment with a chemical com-pound, respectively, with two replicates of each sample:

If you have not already done so, please download the example data that we use in this guide www.decodon.com/Support/Documentation/Delta2D-samples.zip. Unpack the archive. It contains six gel images and match map files for the warping.

Start Delta2D

Start on Windows by clicking on 'Start -> Programs -> DECODON -> Delta2D', on other systems in accordance to the respective installation. Delta2D's Project Manager appears, and you are asked to open an existing project.

Projects are collections of gel images, and analysis data (spot boundaries, quantities etc.). So far, there is only one project, the demo project that comes with the installation of Delta2D.

When Delta2D has started, this dialog provides a list of available projects in the current pool plus the option to change the pool.

Create a new data pool

Delta2D keeps all data related to your current gel images, including the images themselves, in one folder on your hard disk. We call this folder a 'Pool'. It contains several subfolders and data files that are managed by Delta2D. To keep your experimental data organized, we recommend creating a new pool for every new experimental context.

1. Click on the button 'Change Pool' and navigate to a folder where you want to store your new pool.
2. Right click and select 'Create Folder', then click on the new folder or select it and press F2. Type in a name for this new pool and confirm the input with ENTER.
3. Click on OK and confirm the following dialog with Yes.

Choose different pool paths for your different experiments to separate them from each other and from the demonstration data. Furthermore it is easy to copy, move, or backup the different pools. Avoid working with network drives.

Create a new project

The 'Projects' dialog appears again showing an empty list of available projects. The title bar of the dialog displays the path to the pool you are working in. Click on the New button to create a new project. A dialog opens requiring some project details. Provide the name and the author of the project and insert a short description. Do not check the 'Use Internal Standard' checkbox. Press OK to create the new project. Select the new project in the 'Projects' dialog and press Open. Whenever a new project is opened, two new, empty groups are automatically created.

For DIGE projects activate the checkbox 'Use Internal Standard'.

Set up groups for the gel images

It is recommended to organize your gel images into groups. Usually, your project will have one group for every set of replicates in the experiment. We will now create the three groups for our experiment. There are already two groups present, we will simply change their names:

1. Right click on the first group and choose 'Group properties' from the context menu.
2. Replace the group's name by 'control'. If you want, choose another color for the group. Confirm with OK.
3. Repeat this for the second group assigning the name '1min'.

To create a new group, select 'New' from the Group menu. Name it '10min', and choose another color if you want. You can permute, add or delete groups at any time.

Add gel images to the new project

Click on group 'control' to add images to this group. The gel manager opens, showing all gels in the current pool. Since the pool has just been created it is empty and we have to import gel images. Click on the button 'Import' and go to the directory where you un-zipped the example data from our web site. There are sample gel images in a sub folder called 'samples'. Select the two images 'control_01' and 'control_02' by using the left mouse button in combination with CTRL. Click on 'Next'. In this step you can edit names and other descriptive data for an image, and do some basic manipulations such as rotating, mirroring or inverting the image. Click on 'Next' again to repeat this for the second image.

You can click on 'Finish' any time if you want to import the images without further changes. Import the sample images '1min_01', '1min_02', '10min_01' and '10min_02' into their respective groups. Now every set of replicate images is located in a separate group. The project will now appear as in the following figure:

The Project Manager with the six sample gel images organized in three groups ('control', '1min', and '10min'). Every cell represents the respective pair of images.

Setting up a DIGE analysis project

For DIGE and other multiplex experiments, first follow the steps outlined above, i.e. organize gel images into groups according to the sample that they show.
The layout of a simple DIGE experiment with two samples A and B on two gels is shown below. Replicates are put into the same group. The internal standard images are put into the group 'standard'.

Delta2D can apply an internal standard for quantitation. This means that a spot's quantity is divided by the quantity of its corresponding spot on the internal standard image on the same gel. Hence it is important to let Delta2D know which images belong to the same gel and which image shall serve as the internal standard image.

1. Configure the project to use internal standard images
   In the project manager, choose File -> Project properties. Then check the 'Use Internal Standard' checkbox.
2. Assign gels to gel images
   We now have to tell Delta2D which images come from which gel, and where the internal standard images are.
   In the project manager, choose File -> Gel Image Attributes. Select the 'Gel' tab. You see a list of gels (designated by roman numerals) and the images that come from each gel. Rename the existing sample gel names or add new entries assigning new names. Then right click on an image to assign it to a specific gel.
   Alternatively, you can drag an image and drop it onto another gel. Arrange the images to reflect your experimental design.
3. Identify internal standard images
   For each gel, you see a column of radio buttons. Select the radio button next to the image that contains the internal standard (usually, this will be the Cy2 image).