Instructions for Mapping[1] mArray Images using GenePix 5.0
Preliminary Information
· Make sure that the GenePix 5.0 software has been installed on your computer and you have the USB hardware dongle that is compatible with GenePix 5.0
· The tiff images are what one uses to analyze the data from a microarray experiment.
· Before one can analyze these images, it is necessary to create a GenePix Array List (GAL) file (see Howto Create a GAL File.doc). The gal file maps the name of each antigen to the location where it was printed on the slide. This connection is necessary for linking the quantitative information (pixel intensity) to the antigen so that we can make sense of the numbers when we apply statistical techniques to the data set.
· Additional information for using GenePix http://www.axon.com/gn_Genomics.html
Analyzing an Image
Launch GenePix 5.0
A sequence of startup windows will appear the first time one launches GenePix 5.0. Select OK to continue to the next screen. Select Two color mode (traditional) and then OK to continue to the next. Decide whether to receive an update reminder and then select Continue to proceed.
GenePix will complain that it could not open the scanner. That’s fine because we are now going to do an analysis only. Decide whether to view this error every time the program starts then select Analysis Only.
Select the scanner to Emulate. It’s the same model used to scan the mArray slides (most likely 4000B). Decide whether to remember this choice or not so this screen does not appear on the next use of GenePix.
A screen will appear like the one pictured below. I’ve added some tags (shown in red with their keyboard shortcut in parentheses) for the more common buttons or features used in the analysis.
Note that the startup settings can always be adjusted from within GenePix.
Select Options (Alt+I), Other Tab.
Setting the Auto-Alignment Parameters
The auto-alignment tool is key for mapping each slide efficiently. These parameters allow one to automate the spot finding so less manual adjustments are required. The manual adjustments not only take longer, they are a drain on the hands and eyes. Since this step is the necessary evil for processing the data, one might as well do it as efficiently as possible.
Select Options (Alt-I), Alignment Tab.
Set the following parameters on this dialog window to the values suggested below and click OK. Note, these suggestions are good starting points and one may want to use different values depending on spot size, spot location, etc.
· Align features (find circular features)
· Resize during alignment (Min diameter % 66 Max Diameter % 150)
· Max translation (pixels) 10.
Opening an Image
Select File, Open Images… (Ctrl+O).
Open both tiff images (532nm and 635nm) for a particular slide.
Select File, Load Array List… (Alt+Y).
Open the GAL file that was created earlier (see Howto Create a GAL file.doc).
Aligning the Blocks
This step is where the real work begins. It is here that one should spend the most effort because the alignment determines what numerical value of intensity is paired with the antigen name.
Select Zoom Mode (Z). This action allows one to zoom in on a region of interest. Zoom in on the entire slide.
Select Block Mode (B). This action puts one in the position to select and move the blocks. Align the circular (square or irregular) outline within the block to the corresponding set of antigen features spotted on the glass slide.
Left-click the mouse and drag a box around all of the blocks.
Select Align Blocks, Align Features in All Blocks (Shift-F5). This will use the automatic alignment tool on all the features.
Depending on the uniformity of spot sizes, alignment within a grid-like pattern, and background intensity, one may have to adjust individual features within a block.
Select Zoom Mode (Z). Zoom in on a region of interest for a closer look at how the auto-align worked.
Select Feature Mode (F). Move or resize (ctrl-up (enlarge) ctrl-down (shrink)) the features within a block to line up with the spots or to be moved away from spurious spots.
Select File, Save Settings (Alt+S). Save the GPS file for the particular slide in case one needs to re-analyze the slide at a later time.
Analyzing the Blocks and Copying to Excel
With the real work of aligning complete, it is time to have GenePix do some work for us. The analysis tool outputs all the parameters having to do with the spot size, intensity, name, median value, ratio, mean, etc. There are so many parameters GenePix outputs that the results can be overwhelming. Fortunately, we do simple-minded analyses so we only concern ourselves with one or two parameters. I’ll explain those parameters in the next howto installment.
Select Analyze (Alt+A). This step does the real work of converting the pixel intensity of each antigen spot into a spreadsheet format. The analyze step will open up a window like the one shown below.
Note that if you are analyzing a second slide, a window will appear like the one shown below.
As long as you’ve saved the settings as a *.gps file you are free to proceed. Select No and the analysis will go on.
Click on Select All (Ctrl+A) and copy the spreadsheet to the clipboard (Ctrl+C). Note that if you have been transported to the Results tab and one can select the Image tab to return to the image.
Open up Microsoft Excel and Paste (Ctrl+V) the results into excel.
Save this file for later use and continue analyzing the remaining slides. It’s a good idea to include the slide number and print date for the mArray slide. For example, here’s the format I use:
slXX_print_YYYY_MM_DD
[1] To map a mArray image is to associate the antigen names with the spots on the slide. The process of scanning converts the fluorescent intensity of each spot to a numerical value. Now, we have to map or grid these numerical values to their biological name.