Generating Expression Ratios

1. In MAGIC Tool, load the Red and Green image files for OD 6.9. Use derisi_genelist.txt as the gene list. When you begin the addressing step, you can either practice creating a new grid, or open the saved grid 1309.grid.
2. During segmentation, create two different expression files:
   1. Using fixed circle with a radius of 3 pixels, and total signal (without background subtraction) create a new file named my3_10, labeling the column 10 (the number of hours that have passed between OD 0.14 and OD 6.9). Show me how.
   2. Using fixed circle with a radius of 5 pixels, and total signal (without background subtraction) create a new file named my5_10, once again labeling the column 10.
3. Repeat steps 1 and 2 for the OD 7.3 array, with the same settings as above. For the OD 7.3 array, the alternative to creating your own grid for addressing is to use 1313.grid. During segmentation, append the 7.3 data to the 6.9 data in files called my5_last2, and my3_last2. In each file, label the current column 12. Show me how.
4. Now we will see how your data compares to the published DeRisi data.
   1. Use the command Merge Expression Files to combine the two expression files that you have just created, my5_last2.exp and my3_last2.exp, calling the result my_last2.exp (override the default name by simply typing over it). Accept the default nicknames for the two files, which will be appended to the column names. The merge will take a few minutes; you will not be able to open any menus until it is done.
   2. Use the command Merge Expression Files to combine the existing expression file derisi_last2.exp with the merged expression file you just created, calling the result all_last2.exp. Important: you must select derisi_last2.exp as File #1, because all genes in File #1 need to be in File #2 for the merge to work properly.
   3. Log base 2 transform the expression file.
   4. From the Explore window, perform two-column plots comparing your 3 pixel segmentation to the published DeRisi data for the OD 7.3 array (12 hours into experiment), and your 5 pixel segmentation to the published DeRisi data for that same array. Each plot will take a minute or so to appear, so be patient.
      1. Click on an outlier point in one of the plots, turning the point red and causing the ORF name to appear in the bottom right corner.
      2. Go to the other plot, and select the same gene from the drop-down menu in the bottom right corner. Is the ratio in the second plot closer to the published data, or even more different?
      3. Go back to Segmentation in the Build Expression File Menu, which should still contain the OD 7.3 array. Jump to the gene you identified in step (i), and try to explain why the ratio at this particular spot was difficult to determine. Experiment with different segmentation methods to see what you think the best answer is for the ratio at this spot. Answer
      4. As time permits, explore more outliers in the first set of plots, and/or repeat the analysis with the OD 7.3 array.
      5. Explain why it was important to log transform the data before looking for outliers in the two-column plots. Answer
5. Continue to Exploring Expression Ratios

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