Requesting a Ludesi Basic or Pro analysis service

1. Uploading your 2D gel images

After creating a new analysis project , the first step will be to upload the 2D gel images you would like to have analyzed from your computer into REDFIN.

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Navigate to the location on your computer where you have saved the images using the navigation tree on the left [1].

Once you have opened the correct folder the images are automatically uploaded to the panel on the right [2].

Select the images you would like to include and drag them down to the bottom panel [3]. You can also simply double-click on individual images.

REDFIN carries out an automatic image quality check on your 2D gel images, to ensure that they have been captured in the optimal way for image analysis.

If the image capture was not optimal you will receive a warning detailing the problems and suggestions on how to solve them. You then have the choice to go back re-scan the gels or to proceed with the analysis anyway.

Click Next to proceed.

2. Manipulating your 2D gel images (optional)

If any of your images require rotating or flipping in order to bring them into the same orientation as the rest of the images in the project, you are able to easily do this during this step. Similarly, you also have the option to invert images if needed. This is recommended to ensure a more successful analysis further on in the flow.

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Click Next to proceed.

3. Specifying staining type

At this step you only need to distinguish between DIGE staining and non-DIGE staining (single-stains). In case of DIGE experiments, you need to tell the Ludesi Analysis Center which images are internal standards and which are it's "children". This allows us to apply the correct normalization procedure to your 2D gel images.

If you stained your 2D gels using a type of single stain, select the single stain radio button and click Next.

If you are working with DIGE, select the DIGE radio button. The DIGE relationship assignment frame will appear.

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The bottom panel, [1], shows all the images in your project.

Start with dragging the first internal standard image to its designated space, [2].

Drag the corresponding "children" (usually the cy3 and cy5 stained images) to the designated places, [3], underneath that internal standard.

Once all DIGE relations have been defined, click Next to proceed.

4. Defining image relations

If your 2D gel experiment contains technical replicates you have the opportunity to input this information at this step by selecting the Yes radio button.

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The aim is to group together the images that have been run with the same sample (technical replicates). You do this by selecting (or double-clicking on) the first set of technical replicates in the bottom panel [1].

Drag them up into the top panel [2] under the tab Replicate Set.

Click on the Add new tab and repeat the process for every set of technical replicates.

Click Next to proceed.

Note, that images run with the same sample can no longer be considered as being independent from a statistical point of view. By defining them, we are able to apply the correct statistical test to your experiment.

Read more about technical replicates in the blog post "Technical Replicates and Biological Samples"

5. Defining comparisons and groups

At this step you are asked to set up your experimental groups and define how you wish to compare them.

Start by entering the name of the first comparison you wish to make. This could for example be "Control vs Treated". Hit the ENTER key.

Next, enter the name of the first group. For example "Control". Hit the ENTER key.

Drag and drop the images belonging to that group from the bottom panel into the group panel.

You should end up with something like this:

Click on the Add group button to create the next group.

Name the group, assign the gels, and repeat this process until all groups you would like to compare in this comparison are defined.

You can create as many different comparisons as you wish, simply by clicking on the Add comparison button and either creating new groups or selecting existing groups from the drop-down menu. That way you can, for example, compare your samples in terms of condition, as well as time-point, sex, or any other factor.

Once you have set up all your comparisons and groups, click Next to proceed.

6. Defining a region of interest (optional)

In cases where the scanned gel image also includes areas that you do not wish to include in the analysis, you can use the region of interest (ROI) to exclude them.

Such areas are normally located on the periphery of the spot pattern and may include marker ladders, labels, or strong streaking and distortion of the spot pattern.

It is important to note that you only have to define the ROI for one image. The Ludesi analysts will then transfer that ROI to all other images in the most appropriate way. Note, images are NOT cropped during this process.

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If you want to first look through all images in the experiment to decide if an ROI is required, click the Change image button underneath the right panel [1]. Click through all the images in the experiment [1] and select one of the images for drawing the ROI. Click Select image to confirm your choice.

Click on the corner or the edges of the orange rectangle [2] and drag it until it roughly fits the area you wish to have analyzed.

Click Next to proceed.

7. Reviewing your analysis order

Before sending off the analysis order, make sure that your experimental setup and all provided information is correct.

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If you spot any mistakes, simply click the Previous button to go back in the wizard and correct it.

If you are satisfied that all the information is correct, click Next to proceed.

8. Paying for and submitting the analysis order

Depending on the number of gels you wish to have analyzed, the number of required analysis credits is calculated. If you don't have enough analysis credits in your account, you will be prompted to buy more at this stage.

Analysis credits are the currency used in REDFIN to pay for the analysis. One credit is needed per 2D gel analyzed. You can find the current price per credit on our pricing page.

If you have a value code, you can use it at this stage to acquire analysis credits.

Choose if you would like to have your 2D gel images analyzed using the Ludesi Basic or the Ludesi Pro analysis service.

Click on the Buy credits button to purchase the necessary credits, or the Use value code button to enter your value code.

You can read more about buying credits in the "Buying credits and accessing account information" chapter.

Once you have purchased the necessary amount of credits, the Sumbit button will become active. Click it to proceed.

Note, that for security reasons you will be asked to re-enter your password.

Once your experiment has been submitted, the gel images and analysis order information are uploaded to the Ludesi Analysis Center.

As soon as the analysis is completed, the analyzed project is uploaded into your REDFIN account, where you are able to view and work with the data in the REDFIN software.

Next:

Video tutorial: How to work with your results in REDFIN