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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)

Figure 1: Selecting the gene expression workflow

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• Select Library Files… to open the Specify File Locations dialog (Figure 5). This dialog is used to specify the location of the library folder and the annotation files 

PGS will automatically assign the annotation files according to the chip type stored in the .CEL files. If the annotation files are not available in the library directory, PGS will automatically download them and store them in the Default Library File Folder.

• The default library location can be modified at by selecting Change... in the Default Library File Folder panel. By default, the library directory is at C:\Microarray Libraries. This directory is used to store all the external libraries and annotation files needed for analysis and visualization. The library directory can also be modified from Tools > File Manager in the main PGS menu
• Select OK (Figure 5) to close the Specify File Locations dialog

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• A dialog window asking if you would like to save the spreadsheet with the new sample attribute will appear. Select Yes
• Make column 5. (Subject) random by right-clicking on the column header and selecting Properties from the pop-up menu (Figure 9). Select the Random Effect check box from the Properties dialog then select OK. The column 5. (Subject) will now be colored red, indicating that it is a random effect. 
• To save changes to the spreadsheet, select the Save Active Spreadsheet icon (Image Added). Spreadsheets with unsaved changes have an asterisk next to their name in the spreadsheet tree. 

Figure 9: Changing column properties

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Figure 10: PCR PCA Scatter Plot tab

In the scatter plot, each point represents a chip (sample) and corresponds to a row on the top-level spreadsheet. The color of the dot represents the type of the sample; red represents a normal sample and blue represents a Down syndrome sample. Points that are close together in the plot have similar intensity values across the probe sets on the whole chip (genome), and points that are far apart in the plot are dissimilar

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• Another way to see the cluster pattern is to put an ellipse around the Tissue groups. Select Open the Ellipsoids tab on the Plot Rendering Properties Properties dialog and select the Ellipsoids tab
• 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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• Invoke the List Manager dialog by selecting Create Gene List in the Analysis section of the Gene Expression workflow
• Ensure that the 1/ANOVA-3way (ANOVAResults) spreadsheet is selected as this is the spreadsheet we will be using to create our new gene list as shown (Figure 19)
• Select the ANOVA Streamlinedtab. In the Contrast: find genes that change between two categories panel, chooseDown Syndrome vs. Normal and select Have Any Change from the Setting dropdown menu list. This will find genes that have a fold change different between the types of samples
• In the Configuration for “Down Syndrome vs Normal” panel, check that Include size of the changeis selected and enter 1.3 into Fold change >  and -1.3 in OR Fold change <
• Select Include significance of the change, choose unadjusted p-value from the dropdown menu, and < 0.0005 for the cutoff. The number of genes that pass your cutoff criteria will be shown next to the # Pass field. In this example, 23 22 genes pass the criteria. 
• Set Save the list as A, select Create, and then select Close to view the new gene list spreadsheet

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Interestingly, of the 23 genes of the Down_Syndrome_vs_Normal (A) spreadsheet, 20 genes are located on chromosome 21. To save changes to the spreadsheet, select the Save Active Spreadsheet icon (Image Added).

• To create a dot plot showing expression levels of a specific gene for each sample, right click on the row header and select Dot Plot (Orig. Data) from the pop-up menu. This generates a Dot Plot tab for the selected gene (Figure 22)

Image Modified

Figure 22: Dot plot results for gene Down syndrome critical region 3

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Next, we will generate a list of genes that passed a p-value threshold of 0.05 and fold-changes greater than 1.3 using a volcano plot.

• Ensure that the Select the 1/ANOVA-3way (ANOVAResults) spreadsheet in the Analysis tab. This is selected as this is the spreadsheet we will be using to create the gene list
• Select View > Volcano Plot from the PGS main menu
• Set X Axis (Fold-Change) to 12. Fold-Change(Down Syndrome vs. Normal), and the Y axis (p-value) to be 110. p-value(Down Syndrome vs. Normal)
• Select OK to generate a Volcano Plot tab and for genes in the spreadsheet (Figure 23)