Dahlquist:GenePix Pro Software Protocol

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(Gridding and Generating Intensity Data: note that files should be on Desktop)
Current revision (12:34, 30 April 2013) (view source)
(Biology 478/Spring 2013 students:: changed lionshare link)
 
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=== Biology 478/Spring 2013 students: ===
=== Biology 478/Spring 2013 students: ===
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* Follow this link to access your microarray image and .gal files from LionShare:  [https://lionshare.lmu.edu/Users/kdahlqui/BIOL478 https://lionshare.lmu.edu/Users/kdahlqui/BIOL478]
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* Follow this link to access your microarray image and .gal files from [https://lionshare.lmu.edu/xythoswfs/webview/fileManager.action?entryName=/Users/kdahlqui/BIOL478&stk=EDEAEDD3B58C2E2&msgStatus=8%20documents%20have%20been%20successfully%20uploaded%20to%20the%20folder%2C%20BIOL478 LionShare].
* Download both the 532 and 635 wavelength files for your group onto your Desktop.
* Download both the 532 and 635 wavelength files for your group onto your Desktop.
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** '''''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.
* Download the file "Ontario_Y6.4Kv7.GAL" to your Desktop.
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** '''''HINT: Right-click on the link and select the menu item "Save target as" or "Save link as"''''' to download the file.  If you just click on the link, your browser will attempt to open it without downloading
 
=== Gridding and Generating Intensity Data ===
=== Gridding and Generating Intensity Data ===
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* On the right hand side of the screen, select the File icon
* On the right hand side of the screen, select the File icon
** Select Load Array List
** Select Load Array List
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** Red, orange, yellow, green, and pink groups, use the file "yeast_GCAT_Fall2006_gal.gal"
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** Select the file "Ontario_Y6.4Kv7.GAL" (should be on the Desktop)
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** Blue group, use the file "GCAT_384_illumina_Spring2007.gal"
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** Click the Open button
** 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
* 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
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** Click the Normalize button
** Click the Normalize button
* Click on the File icon on the right hand side and select, Save Results As, and save your results.
* Click on the File icon on the right hand side and select, Save Results As, and save your results.
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* Repeat this procedure with the bottom block on the slide.
 
=== Generating an Array Quality Report ===
=== Generating an Array Quality Report ===
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== Looking at the raw data ==
== Looking at the raw data ==
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* We will look at the raw data for two genes of interest, TOD6 (which should have been deleted in this strain), and NSR1, which is a gene known to be induced by cold shock.
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* 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
* Open your results file from the first section of this protocol in Microsoft Excel
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* To find the data for NSR1, first go to the [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=NSR1 TOD6 page at the ''Saccharomyces'' Gene Database] to learn what the systematic name (ID) for NSR1 is.
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* To find the data for ''NSR1'', first go to the [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=NSR1 ''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
* Then search for this ID in your Excel spreadsheet
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* This will tell you the block, row, and column where the NSR1 spot occurs and you can look for it in GenePix Pro.
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* This will tell you the block, row, and column where the ''NSR1'' spot occurs and you can look for it in GenePix Pro.
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* Repeat with TOD6 ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=TOD6 TOD6 page at the ''Saccharomyces'' Gene Database])
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* Repeat with ''SWI4'' ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=SWI4 ''SWI4'' page at the ''Saccharomyces'' Gene Database])
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* We are expecting that NSR1 should be expressed and that TOD6 should not because it has been deleted from the yeast genome.  Is this the case?
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* 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?
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== Next Steps ==
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* Go to the [[BIOL478/S13:Microarray Data Analysis | Microarray Data Analysis]] page for the next steps.

Current revision

Contents

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

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