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***The microarrays contain 29,000 70-mer oligonucleotide elements for Arabidopsis thaliana; these slides were produced at the University of Arizona. More information about the slides, including description of the gene loci, can be found at Platform GPL4570.***

• GOALS of Image Processing
  • Measure intensity of spots/features
  • Quantify gene expression
  • Assess reliability of data
  
  
• Microarray Feature Extraction: Feature extraction is a critical step which greatly influences the outcome of your microarray experiment. Spot-finding programs convert the digital images into numerical values based on the signal intensities of each spot. Most scanners are bundled with automated spot-finding programs based on gal (gene array list) file input.
  
  
• In your lab notebook, include an outline of the image analysis you performed: describe the alignment of the blocks, flagging feature indicators, performing the analysis, generating the scatter plot, etc.

Opening the Image

1. Open GenePix Pro or GenePix Pro Demo.
2. Click the Open/Save button on the right-hand side of the window.
3. Select Open image and click on your image file from the desktop folder.

Loading the Array List

GenePix Pro automatically generates BLOCKS using information contained in a GenePix Array List (GAL) file: these files are used to link the information from the arraying process to the analysis; substance names and identifiers are assigned to each spot (feature).

1. Click the Open/Save button on the right-hand side of the window.
2. Select Load Array List.
3. Go to the Bios 313 folder on the desktop.
4. Open ATv3.5.2.Z_Locus.gal.

Aligning Blocks on an Image

1. Move ALL the blocks to the general area of the array.
2. "Unselect" the blocks.
3. Zoom in and MOVE ONE BLOCK AT A TIME.
   Using the mouse, drag and place each block over a block of spots.
   
   You will have to position EACH block (32 total); for FINE TUNING blocks, see p. 53 in the GenePix Pro User's Guide.
   
   It's OK if some of the BOXES overlap when you're positioning the blocks; what's more important is for the CIRCLES inside the boxes to line up with the SPOTS on the array.
4. Select Feature Mode from the Tools group on the left-hand side of the Image tab; Align Features in ALL BLOCKS.
   *go to "option" ---> "alignment" ---> change to 100 minimum diameter (default = 33%)

Flagging Feature-Indicators

Spend some time looking at the entire slide to flag manually obvious problems, such as:

• smears, streaks, or dust where the spot is the same intensity or lighter than the smear it's sitting in
• individual spots that are misplaced with respect to the grid (sometimes these can be corrected by minor repositioning of the grid)
• spots that have bled into adjacent spots
• anything else that looks "suspicious"

1. To flag a feature, select Feature Mode from the Tools group on the left-hand side of the image.
2. Click on the feature you want to flag.
3. Hit "A" to flag the feature as "Bad."
   
   NOTE: You can also select a region to flag as bad by dragging a "square" around the features.

Saving the Positions of Blocks and Feature-Indicators

1. Click Open/Save on the right-hand side of the window.
2. Select Save Settings As.
3. Enter a file name for the settings and click ok.
   
   Saving this information lets you come back later to perform the analysis.

IMPORTANT NOTE: GenePix Pro uses a local background subtraction method by default; this method is more responsive to background intensity variations across a slide. A different background is computed for each individual feature-indicator, and the median background value is subtracted from the feature intensity before any ratios are calculated (see GenePix Pro User's Guide, pp. 13-15).

Performing the Analysis and Viewing the Results

1. Click on the Analyze button ("BCR").
   
   A JPEG file of the image is saved AUTOMATICALLY.
   
   To SHOW ONLY the UNFLAGGED ROWS, click the Display button in the Table group on the left-hand side of the Results tab and check the unflagged box.
   
2. Switch to the Scatter Plot tab to generate a graph of the analysis measurements for all spots on the array.
3. On the left-hand of the Scatter Plot tab, choose quantities to plot along the X and Y axes:
   • Display data as F1 Median vs. F2 Median: the diagonal through the origin separates features with a higher activity than the control from features with a lower activity than the control.
   • Display data as Index vs Log Ratio: ratio displayed as > or < 1.0 for all spots; differentially regulated features are more easily identified than on a standard intensity comparison plot.
4. Position the mouse over any spot on the scatter plot; the associated feature will be displayed in the Feature Viewer.
   The Feature Viewer reports the intensity of the pixels within the spot, its associated background intensity level, the precise location of the spot in the analysis array, and substance IDs or names that have been defined for each feature.
   
   The Scatter Plot and the Results spreadsheet are fully integrated: after choosing a spot on the scatter plot, select "Group Rows" in the Results spreadsheet; the details about the selected feature will appear at the TOP of the list.

Saving the Data

SAVE YOUR DATA ONCE YOU HAVE COMPLETED THE ANALYSIS.

1. Click the Open/Save button on the right-hand side of the window.
2. Select Save Results As.
   NOTE: The default file format is *.gpr.
3. Name the file and click OK.
4. Next, save your customized view of your Results:
   a. Click the Open/Save button.
   b. Select Export Results.
   c. Name the file and click OK.