Dahlquist:GenePix Pro Software Protocol: Difference between revisions
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== Using GenePix Pro == | == Using GenePix Pro == | ||
=== Biology 478/Spring 2013 students: === | |||
* 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. | |||
** '''''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 === | === 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 File > Open Images | ** Select File > Open Images | ||
** Navigate to the folder containing your .tif 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 | ** Hold down the Control key while clicking to select both the 532 and 635 wavelength files | ||
** Click on the Open button | ** Click on the Open button | ||
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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 | ||
** | ** Select the file "Ontario_Y6.4Kv7.GAL" (should be on the Desktop) | ||
** 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. | ||
=== Generating an Array Quality Report === | === Generating an Array Quality Report === | ||
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* Name the file: Chip #_Year-Month-Date | * 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 | * 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 [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 | |||
* 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'' ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=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 [[BIOL478/S13:Microarray Data Analysis | Microarray Data Analysis]] page for the next steps. |
Revision as of 10:34, 30 April 2013
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
- Go to the Microarray Data Analysis page for the next steps.