Dahlquist:GenePix Pro Software Protocol
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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.
- Save images as "Single-image TIFF Files (*.tif)". Do not use TIFF LZW compression (lossless).
- Name files with a Date prefix and Barcode prefix.
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
- Go to the Microarray Data Analysis page for the next steps.