Chapter 1
Analyzing Gel Images with Delta2D

The next chapter will explain the usage of Delta2D at the example

1.1 Setting up Projects

Normal Experiment with Replicate Groups

________________________________________________________________________________

Note:

Based on image warping and image fusion, DECODON has introduced full expression profiles to avoid missing values in the quantitation table and the resulting problems during statistical analysis. To receive full expression profiles, create a Proteome Map make an union fused image out of the whole set of images in your experiment(at least out of one representative image of every group of replicates). Then do the spot detection on the resulting Proteome Map onlyand transfer the spots to the original images.

Read more about the benefits of 100% per cent Spot Matching at www.decodon.com/Solutions/Delta2D/100_Percent_Spot_Matching.html.

Create a New Pool

To keep our experimental data handy, we recommend to create a new pool for every new experimental context. (See section 4.1 for details on how to do this)

Import Your Gel Images Into Delta2D

See section 4.1

Load the images into your project

Create a new Project (see 4.2), include the relevant images and distribute them on groups, so that replicate gel images from the same sample are all in the same group.

Assign the Group Warping Strategy

Use the Strategy Manager (See section 5.10) to assign the Group Warping Strategy to the complete project. It might be necessary to change the warp mode for single pairs, but using the warping manager you make sure to obtain persistent warping chains and don't produce warping cycles. Additionally you reduce the number of gel pairs you have to handle to the minimum necessary explicit warps.

Edit the explicit warps

Examine the gel image pairs with explicit (any warp mode but the implicit one) warp mode one by one and correct them where necessary by setting match vectors and / or even changing the warp mode to a more suitable if necessary. For details please refer to section 5.7.

Compensate for Experimental Variations

Depending on the number of replicates per group, you might want to condense the replicate groups to a single image per group, compensating for all experimental variations within each group. To achieve this, create an Fused Image of each group, using average fusion. In section 4.8 you can learn everything about image fusion and how to perform it.

Create a Fused Image of your complete project

Irrespective whether you produced Average Fused Images from your groups, it is a good idea to create a fused image over the complete project, using either Max Intensity or Union as fusion type. Please refer to section 4.8 for details, or simply produce both image fusions and compare them, if you don't know which one to prefer. When doing so, please be careful not to include the first fused image when producing the second.

Detect Spots

on the image fusion to include every spot existing on any gel image in your project. Thus you get a central set of spots, which can be edited on a central place and and transferred to the other gel images afterwards. If necessary you can adjust parameters to reduce the number of false positive or increase the number of detected weak spots. Details on detection parameters can be found in section 6.1.

Edit Spots

to separate two spots detected as one or to join two parts of a spot detected accidentally as separate spots if necessary. Spot editing needs to be done on your "master" set of spots, if you plan to transfer spots.

You can find more on editing spots in section 6.5.

Transfer Spots

to all other gel images like described in section 4.9.

Open Quantitation Tables

to view the quantitation of all spots. Sort the tables after your needs to find the interesting spots or refer to the next section on how to identify the interesting spots.

1.2 Finding Interesting Spots

Identifying Interesting Spots by their Appearance

Finding Interesting Spots in a Scatter Plot

Figure 1.2: Scatter plot: illustrates the expression differences between two gel images

Identify Interesting Spots by Their Expression Profile

You can even see an expression profile of a spot over the complete project: in the menu of the Dual View, please click on Rollups  Expression Profiles. A new window will open, keeping its position in front of its parent window: a so-called Rollup. Make sure that the Segments Tool is selected and simply point to any spot. You don't need to click or select it in any way, just keep the mouse cursor above it. Now move the mouse pointer to the next spot and watch the Expression Profiles Rollup. It changes its display, showing the expression of the respective spot across the complete project.

Figure 1.3: Expression profil rollup indicating project wide spot intensity

Filtering for Interesting Expression Profiles

Switch to the Quantitation Table by choosing from any menu Window  Quantitation Table. What you see now is a set of tables full of values. You have a lot of data but still don't feel informed. How we can find relevant spots now? Delta2D offers powerful tools to obtain your results: arbitrarily combinable filters. With an adequate combination of filters, you can obtain the set of spots corresponding to nearly every question. Let's try it out: We are looking for spots matching the following criteria:

• increased intensity by a factor of at least 2²
• increase should be observable under experimental conditions used in all images compared to the control image

To see only the spots matching these criteria, just take the following steps:

1. Switch to the quantitation table and select table for allimages.
2. Make sure that the control image is selected as the Ratio Master in the Table Properties window. Use the menu item Column Table Properties to find this setting.
3. Find the first column containing Ratio data. To see the name of a column, either drag the line between two column headers to make it wider, or simply point to its header and wait a second. Its name will appear as a tool tip (do not click). Click on the top part of the column header, labeled Filter to open the respective filter.
4. Find the Label in the section above and click on the field below this label.
5. Type 2into the field Show values from:, or drag the left slider below the histogram in this dialog to the right until the value of this field gets as close to 2 as possible.
6. The check box Filter active will be checked automatically since you have changed the filter settings.
7. Apply the settings with the button Apply.
8. Click now on the next ratio column in the table and repeat the above steps 4-7.
9. Repeat the procedure with each ratio column in the statistics table.

Switch back to the Dual View to see the spots matching these criteria.