Dahlquist:GenePix Pro Software Protocol

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Revision as of 09:34, 30 April 2013 by Kam D. Dahlquist (talk | contribs) (Biology 478/Spring 2013 students:: changed lionshare link)
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Using GenePix Pro

Biology 478/Spring 2013 students:

  • Follow this link to access your microarray image and .gal files from LionShare.
  • Download both the 532 and 635 wavelength files for your group onto your Desktop.
    • HINT: Right-click on the link and select the menu item "Save to Desktop" to download the file.
  • Download the file "Ontario_Y6.4Kv7.GAL" to your Desktop.

Gridding and Generating Intensity Data

  • Launch GenPix Pro (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 (should be the Desktop)
    • 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" (should be on the Desktop)
    • 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
    • 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)
  • In the Results tab, click on the button to Configure Normalization
    • Make sure that "Ratio-based" is selected and it says that the "Ratio of medians" of "all of the features" is equal to "1"
    • Click the Normalize button
  • Click on the File icon on the right hand side and select, Save Results As, and save your results.

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