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5 guidelines for getting the best 2D gel image analysis results from REDFIN Solo
Posted by ludesi in 2DE Knowledge Base, How to's in REDFIN, Tips and Tricks
Please note that the following post only applies to REDFIN Solo. If you are using our Basic or Pro offerings, the guidelines are not applicable, as a different analysis strategy is employed in these cases.
REDFIN Solo is a powerful tool to achieve great 2D gel image analysis results, but like with all things you need to use it in the right way to get the most out of it.
So straighten your reading glasses and move that little bit closer to the screen because here are five tips that you don’t want to miss out on if you are using REDFIN Solo for your 2D gel image analysis:
1. Choose a good warp reference image
In the first step of the REDFIN Solo analysis wizard you are asked to select a warp reference image. As all other images in your experiment will be warped (a form of alignment) to this one gel image it is important that you choose the most suitable one. A suitable warp reference image is basically the nicest looking, most consistent gel image.
Flip through all the images in your experiment using the image selector (black field containing thumbnails of all your images) and look for the image that has the least streaks and distortions, whilst at the same time shows a clearly separated spot pattern. Once you have selected a suitable warp reference image you are already off to a good start in the analysis.
2. Warping, warping, warping
The most challenging but also the most important step in the analysis workflow. The better you manage to warp the images to each other, the better your results will be, so it is worth spending some extra time on this one.
Start off with an auto-warp. Generally you don’t need to do anything before hitting the auto-warp button, but if the spot patterns of the images are very displaced from the reference image you can help the auto-warp by simply adding one or two manual warp anchors before auto-warping.
Setting a good region of interest (ROI) for the auto-warping has a bigger effect on the success of the auto-warp as you may think. Generally speaking, it is better to have the ROI a little too small (not including all spots along the borders) than too large (including all spots but also some noise). More specifically, make sure not to include anything outside of the spot pattern, such as part of the scanner bed, labels, the dye front or marker ladders, as they could cause the auto-warping to partly fail. Similarly, if you have a lot of streaking along the edges of the gel you are well advised to exclude them as much as possible.
The warping ROI should only include the spot pattern.
After running the auto-warp take a few minutes to check the quality of the warping in every image. A good way to do this is to click on the warp animation thumbnail to enlarge it and flip through all gel pairs. Using the warp animation watch out for any areas with lateral movements as they are an indication of un-warped areas (although careful of PTM’s as they should often remain un-warped).
If you see individual spots or small areas that need tweaking, you can quickly fix those by placing manual warp anchors (and possibly removing the auto-warp anchors in that region).
If for some reason you are generally not happy with the results of the auto-warping, remove all auto-warp anchors by hitting the button “Delete all automatic warp anchors in current pair”. This will remove only the automatically placed anchors (red crosses) whilst leaving the manual warp anchors (red circles) in place. Set a handful of manual anchors and then run the auto-warp again.
You should now be left with a good warping in all of your gel images. If not, repeat the cycle – the time spent will be worth it!
3. Exclude strange gels from the fusion image
REDFIN Solo uses a 4th generation workflow, which means that it creates a fusion – or composite – image of all the warped images, spot detects on this fusion image, and then pushes the resulting spot borders back onto all individual images.
It is therefore important to have the option to exclude “bad” gel images from the fusion image and this is exactly what you can do in the third step, Fusion, of the analysis workflow. A “bad” gel image in this context can be described as any gel that has larger cracks, bubbles, or any other larger artifact, or that shows strong distortions in the spot pattern that make it very different from the rest of the gel images. These images are usually also difficult to warp.
If you don’t exclude such gel images from the fusion image, these distortions can cause unsatisfactory spot detection further on in the workflow.
Please note, that by excluding a gel image from the fusion image you are not excluding it from the overall analysis! The generated spot borders will also be pushed back to gel image that has been excluded from the fusion image.
4. Be thorough when you check the spots
At the Spots step in the workflow it might be tempting to only give it a quick glance before accepting the settings and clicking “Next”. However, as with the warping step, a few extra minutes spent here will potentially save you a lot of time later on.
This is the point where you are able to globally edit the spot detection in the entire project. By working in only the one image you will improve spot detection in every single gel of your experiment. It is good practice to not only adjust the “Number of spots” slider to an appropriate level, but to also zoom into the gel using your mouse wheel and removing/adding individual spots by right and left clicking the mouse. Again, a few minutes work that can greatly improve the results later on.
5. Make sure borders are big enough
As a general rule of thumb, it is better for the spot borders to be a little too big than a little too small, as it makes them more forgiving of small warping mistakes. At the Borders step in the Solo workflow, make sure that you enhance the image contrast and possible darken the gel a little bit in order to see the full spot outlines. Then adjust the border size slider until the borders encompass all of the spot signal.
If you follow these 5 guidelines you should be able to achieve very high quality image analysis results on your 2D gel projects. If you are unsure about what to do in the different steps of the analysis wizard, make sure to read the directions on the right hand side and watch the video tutorials.
For questions or suggestions feel free to get in touch with us – we love to hear from you! 
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