Dahlquist:GenePix Pro Software Protocol

Using GenePix Pro

Scanner Settings

• Version information for the GenePix Pro software used to scan chips in the Dahlquist Lab.

• Hardware settings:
  • We use a pixel size of 10 microns.
  • We use two lines to average.
  • We use a focus position of 0.
  • We use Auto PMT settings at 100% power (see below).

• Auto PMT settings:
  • Saturation tolerance is set to the default value of 0.05%.

• Alignment Options:
  • Find irregular features
  • Resize features during alignment to minimum diameter 33% and maximum diameter 200%
  • Flag features that fail background threshold criteria as Not Found
  • Estimate warping and rotation when finding blocks
  • Automatic Image Registration: Max translation 10 pixels.
• Saving scanned images:
  • Create a folder on the C: drive called "MicroarrayData_yyyymmdd" where yyyymmdd is the current date.
• Save images to this folder as "Single-image TIFF Files (*.tif)". Do not use TIFF LZW compression (lossless).
  • Name files with a Date prefix and Barcode prefix.

Gridding and Generating Intensity Data

• Launch GenPix Pro 6.1 (select Analysis Only)
• On the right hand side of the screen, select the File icon
  • Select File > Open Images
  • Navigate to the folder containing your .tif images
  • Hold down the Control key while clicking to select both the 532 and 635 wavelength files
  • Click on the Open button
• Use the brightness and contrast sliders on the upper left hand side to adjust your image to see all of the spots
• You can use the magnifying glass icon to zoom in on an area of the image, and the magnifying glass return icon to go back to the whole chip image
  • Zoom in so that you can see the entire top block of grids on your screen
• On the right hand side of the screen, select the File icon
  • Select Load Array List
  • Select the file Ontario_Y6.4Kv7.GAL
    • To download this file, right-click on the link and select "Save link as..." from the context menu that appears.
  • Click the Open button
• Using the icon that has an arrow and a square, drag the grids so that they are approximately aligned with the top block of spots
  • Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array, Find All blocks, Align Features
    • Alternately, you can do this in three steps by:
      • Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array
      • Click on the icon again, and select Find All Blocks
      • Click on the icon again, and select Align Features in All Blocks
• On the right hand side of the screen, select the icon that looks like a table of data (it says B, C, R at the top of it)
• Click on the File icon on the right hand side and select, Save Results As, and save your results as type "GenePix Pro Files (*.gpr)". Save a JPEG image containing all analyzed features.

Generating an Array Quality Report

• Launch GenePix Pro (select Analysis Only)
• On the right hand side of the screen, select the File icon
• File > Open Results
• Select the desired file
• Select Report tab at the top of the screen
• On the left side of the screen, under Navigate, select the back arrow or home icon
• Select Array Quality Control
• Adjust Vital Statistic Thresholds values to:

Quality Control Limits
Median signal to background > 2.5
Mean of Median background < 500
Median Signal to Noise	> 4
Median % >B + 1 StdDev	> 90
Feature variation < 0.5
Background Variation < 1.2
Features with Saturated pixels	< 3.3%
Not Found < 18%
Bad Features < 7%

• Select Start
• Select Show Printable Version
• File > Print
• Under Select Printer > Select Adobe PDF
• Select Print
• Save on the Desktop
• Name the file: Chip #_Year-Month-Date
• The PDF file will either open on its own or you need to open it from the file you saved it to

Looking at the raw data

• We will look at the raw data for two genes of interest, SWI4 (which should have been deleted in this strain), and NSR1, which is a gene known to be induced by cold shock.
• Open your results file from the first section of this protocol in Microsoft Excel
• To find the data for NSR1, first go to the NSR1 page at the Saccharomyces Gene Database to learn what the systematic name (ID) for NSR1 is.
• Then search for this ID in your Excel spreadsheet
• This will tell you the block, row, and column where the NSR1 spot occurs and you can look for it in GenePix Pro.
• Repeat with SWI4 (SWI4 page at the Saccharomyces Gene Database)
• We are expecting that NSR1 should be expressed and that SWI4 should not because it has been deleted from the yeast genome. Is this the case?

Next Steps

• Go to the Microarray Data Analysis page for the next steps.