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The whole is simpler than the sum of its parts.
Willard Gibbs

Scanning the Microarray

The GenePix 4000B Microarray Scanner is in Biology B03 (basement lecture room).
The scanner must be running before the computer is powered up; the instructor will turn on the scanner in advance so it warms up for at least 15 minutes.

Cy3 is excited by GREEN laser light (532 nm); Cy5 is excited by RED laser light (635 nm). If the control sample is labeled with Cy3 and the experimental sample with Cy5, then a RED spot indicates that the experimental sample for this feature is MORE abundant than the control sample; a GREEN spot indicates that the experimental sample is LESS abundant; a YELLOW spot means that there is NO CHANGE in the level between the two samples.
  1. Open the GenePix Pro 4.0 software.
  2. Slide open scanner door.
  3. Insert the MA slide in the holder, ARRAY-SIDE DOWN.
  4. Open the Hardware Settings box: click on the picture of the scanner with a wrench on the right-hand side of the window.

    NOTE: Genisphere Inc. (3DNA Array 900 Kit) recommends the following initial scanner settings:
    *for Cy3, laser power at 100, PMT gain at 500-700 volts
    *for Cy5, laser power at 100, PMT gain at 600-800 volts

  5. Click on the Preview Scan button (2 arrow points) on the right-hand side of the window.
    *The preview scan is performed at low resolution (40 µm) to find the area on the array that you want to scan at high resolution.

    While performing the preview scan, zoom in to the part of the image that contains the features:
    a. Switch to the histogram tab.
    b. Make sure the Min and Max Intensity cursors are set to 500 and 65530, respectively.
    c. Adjust the PMT Gain in each channel until this ratio is about 1.0. The ratio of counts in each channel is reported at the bottom left of the histogram.
    DO NOT ADJUST THE GAINS DURING A DATA SCAN.

    NOTE: if present, the BACKGROUND should be more YELLOW.

  6. After the preview scan, select the Scan Area tool (rectangle in a black box) from the Tools group on the left-hand side of the Image tab.

    Hold down the mouse and draw a rectangle around the region containing the features; this area ONLY is scanned during a Data Scan.

  7. In the Hardware Settings box, change the Lines to Average to 2 (the scanner scans each line twice and averages the value).
  8. Click on the Data Scan button (1 arrow point) on the right-hand side of the window.

    At the end of the scan, record the PMT gain for each channel and the OVERALL ratio. An ideal ratio is approximately 1 (0.9-1.1); if the OVERALL ratio seems low, zoom in on a nice area of the array and check the ratio there.

    Examine the scanned image immediately to determine the number of elements that are low intensity or are saturated ("white" spots); the proportion of these elements should be quite low since information is lost in both cases. You may need to rescan if the proportion of such elements is large; although the absolute value of the spots may be reduced by repeated scanning(s), the relative fluorescence is preserved, so information is not lost.

  9. SAVE the image(s) of the scanned array as a multi-image TIFF file:
    a. Click the Open/Save button on the right-hand side of the window.
    b. Select Save Images.
    c. Select Multi-image TIFF Files.
    d. Name the image (include brief experimental notation, the date, PMT settings, and overall ratio) and click Save.

    After scanning the array, you MUST SAVE the images; analysis can be done at any time later.

  10. Put the MA slide into a light-protective slide box.

    Save your image files to the "BIOS 313 SPRING 2006" folder on the desktop.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 12 July 2002
Updated 7 March 2007