Now that you have obtained statistical results from the microarray experiment, you can create new spreadsheets containing just those genes that pass certain criteria. This will streamline data management by focusing on just those genes with the most significant differential expression or substantial fold change. The List Manager can be used to specify numerous conditions for selecting genes of interest. In this tutorial, we are going to create a gene list of gene with a fold change between -1.3 to 1.3 that has an unadjusted p-value of < 0.0005.
This will find genes with different expression levels in the different types of samples.
The number of genes that pass your cutoff criteria will be shown next to the # Pass field. In this example, 30 genes pass the criteria.
The spreadsheet Down_Syndrome_vs_Normal (A) will be created as a child spreadsheet under the Down_Syndrome-GE spreadsheet.
This gene list spreadsheet can now be used for further analysis such as hierarchical clustering, gene ontology, integration of copy number data, or be exported into other data analysis tools such as pathway analysis.
You can practice creating new gene list criteria of your own to become familiar with the List Manager tool. For more information, you can always click on the () buttons.
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.
In the plot, each dot represents a gene. The X-axis represents the fold change of the contrast (Down syndrome vs. Normal), and the Y-axis represents the range of p-values. The genes with increased expression in Down syndrome samples are on the right side of the N/C (no change) line; genes with reduced expression in Down syndrome samples are on the left. The genes become more statistically significant with increasing Y-axis position. The genes that have larger and more significant changes between the Down syndrome and normal groups are on the upper right and upper left corner.
In order to select the genes by fold-change and p-value, we will draw a horizontal line to represent the p-value 0.05 and two vertical lines indicating the –1.3 and 1.3-fold changes (cutoff lines).
The plot will be divided into six sections. By clicking on the upper-right section, all genes in that section will be selected.
Note: If no column is selected in the parent (ANOVA) spreadsheet, all of the columns will be included in the gene list; if some columns are selected, only the selected columns will be included in the list.
The description is shown when you right-click on the spreadsheet > Info > Comments. Here, I have named the list "volcano plot list" and described it as "Genes with >1.3 fold change and p-value <0.05" (Figure 14). 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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