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For this tutorial, unzip the files to C:\Partek Training Data\Down_Syndrome-GE or to a directory of your choosing. Be sure to create a directory or folder to hold the contents of the zip file
Copy or move the annotation files (HG-U133A.cdf, HG-U133A.na36.annot, HG-U133A.na36.annot.idx) to C:\Microarray Libraries. (Copying the annotation files to the default library location is done because newer annotation files that are released after the publication of this tutorial may cause the results to be different than what is shown in the published tutorial. If, however, you prefer to download the latest version, you may omit copying the HG-U133A files to C:\Microarray Libraries)
Start PGS and select Gene Expression from theWorkflowspanelon the right side of the tool bar in the PGS main window (Figure 1)

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	Figure 1
	Figure 1

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Figure 1: Selecting the gene expression workflow

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Open the Plot Rendering Properties dialog and select the Ellipsoids tab (Figure 13)
Select Add Ellipse/Ellipsoid
Select Ellipse in the Add Ellipse/Ellipsoid... dialog
Double click on Tissue in the Categorical Variable(s) panel to move it to the Grouping Variable(s) panel
Select OK to close the Add Ellipse/Ellipsoid... dialog and select OK again to exit the Plot Rendering Properties dialog

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	Figure 13
	Figure 13

Figure 13: Adding Ellipses to PCA Scatter Plot

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To invoke the ANOVA dialog, select Detect Differentially Expressed Genes in the Analysis section of the Gene Expression workflow
In the Experimental Factor(s) panel, select Type, Tissue and Subject by pressing <Ctrl> and left clicking each factor
Use the Add Factor > button to move the selections to the ANOVA Factor(s) panel
To specify the interaction,select Type and Tissue by pressing <Ctrl> and left clicking each factor. Select the Add Interaction > button to add the Type * Tissue interaction to the ANOVA Factor(s) panel (Figure 16). Do NOT select OK or Apply. We will be adding contrasts to this ANOVA in an upcoming section of the tutorial.

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	Figure 16
	Figure 16

Figure 16: ANOVA configuration

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Select Add Contrast to add the Down Syndrome vs. Normal contrast
Select OK to apply the configuration
If successfully added, the Contrasts… button will now read Contrasts Included
By default, the Specify Output File is checked (Figure 17) and gives a name to the output file. If you are trying to determine which factors should be included in the model and you do not wish to save the output file, simply uncheck this box
Select OK in the ANOVA dialog to compute the 3-way mixed-model ANOVA
Several progress messages will display in the lower left-hand side of the ANOVA dialog while the results are being calculated.

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	Figure 17
	Figure 17

Figure Figure 17: Configuring contrasts for ANOVA

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In the Down_Syndrome_vs_Normal (A) spreadsheet, right click on the second column header 2. ProbesetID and select Insert Annotation from the pop-up menu (Figure 23)
Select Chromosomal Locationunder the Column Configuration panel. Leave everything else as default and select OK

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	Figure 23
	Figure 23

Figure Figure 23: Adding a gene annotation

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Select Rendering Properties ()
Choose the Axes tab
Select the Set Cutoff Lines button and configure the Set Cutoff Lines dialog as shown (Figure 26)
Check Select all points in a section to allow PGS to automatically select all the points in any given section
Select OK to draw the cutoff lines
Select OK in the Plot Rendering Properties dialog to close the dialog and view the plot

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	Figure 26
	Figure 26

Figure 24: Setting cutoff lines for -1.3 to 1.3 fold changes and p value of 0.05

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The list can be saved as a text file (File > Save As Text File) for use in reports or by downstream analysis software.

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	Figure 27
	Figure 27

Figure 27: Creating a gene list from a Volcano Plot

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PGS Documentation