Analyzing your gels in REDFIN

1. Uploading your 2D gel images

After creating a new analysis project you will be expertly guided through the analysis flow step-by-step. The first step will be to upload the 2D gel images you would like to include in the analysis 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 software which images are internal standards and which are it's "children". This allows REDFIN 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. Selecting a warp reference image

Now that you have successfully set up your project, the actual analysis workflow starts. The 4th generation analysis strategy of REDFIN is based on image warping and spot detection in a fusion image. A fusion image is a composite image of all images in your project. The spots detected in the fusion image are then pushed back to all individual images.

The first step is therefore to detect a suitable reference image to be used for subsequent warping. Warping means alignment of all images to each other using special algorithms.

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Flip through all the images by clicking on the thumbnails in the image selector [1]

Identify the image that appears to have the most consistent spot and the least distortions, smears, or other arterfacts. As this image will be used as a template to which all other images will be warped, you want to make sure to chose the "best" one.

If you have decided which image to use as the warp reference click on the OK button [2]. The warp reference image receives a green border and will subsequently be indicated by the color green.

Click Next to proceed.

5. Aligning all your images using warping

Good warping of your images is one of the main factors ensuring good image analysis results at the end. Although REDFIN will auto-warp all your images for you, it is good practice to check the success of this automatic alignment at completion. Especially proteins on the periphery of the gels tend to run inconsistently and the warping in these areas may require manual tweaking.

The warping interface in REDFIN is described below:

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[1] The warp area shows the reference image (green) overlaid with the image that needs to be warped to the reference (magenta).

[2] In the black image selector area you can click on the different images to flip through the warp pairs.

[3] The image visualization controls allow you to adjust brightness and contrast for the green and magenta colored images, enabling you to visualize also fainter spots.

[4] The image navigation controls allow you to zoom in and out of the warp pairs, as well as move around in them. Note, that you can also use the scroll wheel of your mouse to zoom, as well as the combination SPACEBAR + mouse pointer to move around the image. Click on the center square to reset the image.

[5] The correlation trend indicates if a placed warp anchor has improved the warping or not. The closer the correlation coefficient is to 1 the better the warping. A green arrow indicates an improvement, whereas a red arrow pointing down indicates a worsening of the warping. The correlation trend should however only be used as a rough guideline. A correlation of 1 is more or less impossible to achieve.

[6] The warp animation is ideal for assessing the quality of the warping. Click on the thumbnail to enlarge the animation. Lateral movement is an indication of areas that may need improvement. Pulsating spots on the other hand merely indicate spots with differential spot volumes and require no further adjustment.

[7] The analysis tools include a button for initiating the auto warp wizard, as well as button that will delete all automatically placed warp anchors in the currently selected image.

Recommended warping workflow:

Step 1. Flip through all the gel images in the image selector and check for any who's spot pattern is strongly shifted away from the reference image. In those cases it is recommended to place one or two manual warp anchors to bring them into closer alignment. You place manual warp anchors by left clicking anywhere within the warp area, releasing the mouse button, dragging the magenta image on to the equivalent area of the green reference image, and releasing the mouse button again. You can delete any anchors by right clicking on them.

Step 2. Click on the Auto-warp button under the Analysis Tools to initiate the auto warp wizard.

Make sure that the warp area encompasses only the spot pattern. It is recommended to make the blue rectangle slightly tighter than the outermost spots, in order to not include areas of high variability as well as streaking commonly found on the periphery of the gel image.

Click Next.

Unless you wish to first try the auto-warp on one warp pair only, select the All gels radio button and click Run auto warp to initiate auto warping on all images in the experiment.

Step 3. Once the auto warp is completed you are able to check the result on all images by clicking through the thumbnails in the image selector. For better assessment of the warp quality click on the animation thumbnail to open the animation window.

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Automatic warp anchors are indicated by red crosses.

You will also notice that each image thumbnail in the gel selector now has a blue background (indicating the presence of warp anchors in that image) as well as a number attached indicating how many warp anchors have been placed.

Step 4. For any areas in the gel image that require improving in the warping, add manual anchors by clicking and dragging. Manual anchors are indicated by red circles.

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If the warping has failed in an area (indicated by strong distortions in the image), a good strategy is to first delete all warp anchors in that area and proceed to manually warp that area.

Step 5. Once you are satisfied with the warping click Next to proceed.

6. Excluding images from the fusion image (optional)

A fusion image is a composite image of all images in the project. It contains all spots present in this experiment and can be considered as a "master gel". Spots are detected on this fusion images and later pushed back onto all individual images in the experiment, ensuring 100% matching and no missing values.

This so-called 4th generation analysis strategy also enables global spot editing on the fusion image, which saves a lot of time later on in the workflow.

All you need to do at the fusion image step is to quickly flip through the gel images again and make sure that there are no images that display large artefacts, such as cracks or big bubbles. It may be advisable to exclude such images from the fusion image, as they can introduce distortions, which interfere with spot detection. You exclude gel images by unchecking them.

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In extreme cases, when your experiment includes gel images that differ strongly from the rest of the images, it may also be advisable to exclude them.

Note, that any images that are excluded will still be analyzed!

Click Next to proceed.

7. Optimizing spot detection

REDFIN automatically detects spots on the fusion image, which are indicated at this stage by red dots.

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It is recommended to spend some minutes checking the spot detection, to make sure the optimal results has been achieved for your experiment.

Start by adjusting the contrast and image brightness to visualize also fainter spots.

Play around with the slider [1] until you feel that a good balance has been achieved between detecting fainter spots without over-detecting.

Add individual spots by left clicking on the spot center. Similarly, you can delete any incorrect spot by right clicking on it.

For some images you may want to draw an area of interest to exclude artefacts and badly resolved spots in the peripheral area of the gel. You can do this by clicking on the Draw area button, and clicking sequentially around the image to draw the area you deem adequate.

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When you are satisfied with the results click Next to proceed.

8. Optimizing spot borders

REDFIN automatically creates spot borders for all the spots detected in the previous step.

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Check if the border size is optimal for your gels, by adjusting the contrast and brightness settings until you have visualized also fainter spots.

If necessary, adjust the border size using the slider [1].

The changes are first only applied to the preview area underneath the slider. Once you have found a setting that is suitable for the majority of spots, click the Apply button to affect the entire gel image.

Click Next to proceed.

9. Reviewing the image analysis results

Before you are required to pay for the analysis by purchasing analysis credits, you are able to review the results on all individual gel images. That way, if you are not happy with the image analysis, you are able to abort the analysis without paying, and you won't spend money on results you cannot use.

You can also exclude individual gels from the analysis at this stage, simply by un-checking the orange tick mark on that gel. You might want to exclude gels if the quality of the spot pattern was not good enough to allow an acceptable spot detection or matching.

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Check the image analysis results on all individual gel images by clicking through the image thumbnails in the image selector [1].

To exclude individual gels from the analysis, simply un-check the orange tick mark on that gel [2].

If you are happy, click Next to proceed.

10. Purchasing analysis credits

If you don't have analysis credits in your account, you will be prompted to buy more at this stage. Analysis credits are the currency REDFIN uses 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.

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Click on the Buy credits button [1] to purchase credits, or the Use value code button [2] to enter your value code.

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

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

Up until this point, any data has only been saved locally to the computer you are working on. However, from now on all data is also continuously backed-up to Ludesi's servers, allowing you to access it from any computer in the world.

11. 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, REDFIN is able to apply the correct statistical test to your experiment.

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

12. Defining comparisons and groups

In the final step of the analysis wizard, you will 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.

You are now taken to the Results Viewing Environment in REDFIN, where you are able to identify differentially expressed protein spots of interest.

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Next:

Five guidelines for getting the best 2D gel image analysis results from REDFIN