TNF-mediated gene expression in HUVEC

The following tutorial walks through the identification of genes regulated by TNF in HUVEC (Human Umbilical Vein Endothelial Cells). This analysis showed that genes involved in distinct biological processes, including immune response, cell cycle, apoptosis and cell adhesion, are regulated by TNF in HUVEC.

Before you begin, we recommend that you review the analysis summary. The top button of the four on the right will download it if you haven't already.

When you are ready to begin this hands-on tutorial, click the third button down on the right, labeled "Log in to the dataset with tutorial."

1. Select Pairwise in the menu bar on the left side of the screen labeled Control Panel. You will see “Pairwise” under the heading marked “Analysis.”

2. Select the magnifying glass icon next to “U133A Human Genome” in the list. There are approximately 20,000 transcripts represented on this array. For this analysis, GC-RMA was used to derive expression measurements from the probe-level (CEL file) data.

3. Pairwise analysis is used to identify differentially expressed genes in two groups. There are four replicates for each of the two groups in this study. Select the four replicates for the untreated HUVEC samples (HUVEC) to place them in Group 1.

4. Select the four replicates for the TNF treated HUVEC samples (HUVEC + TNF) for Group 2.

5. Pairwise analysis combines a fold-change cutoff with a quality filter and comparison statistics to generate a list of differentially expressed genes. Select the following settings:

Normalization: None
Data was normalized previously.

Statistics: t-test
Performs a two-sample, unpaired t-test for each gene that passes the quality and fold-change cutoffs.

Quality: N/A
GC-RMA does not generate detection calls.

Threshold: Lower = 1.5; Upper = None.
Filters out genes with less than a 1.5 fold change in expression.

Correction: Benjamini and Hochberg
Calculates a false discovery rate from the raw p-values using the method of Benjamini and Hochberg.

Data transformation: Data Already Log Transformed
Data was log base2 transformed during GC-RMA transformation.

6. Select the Analyze button.

7. After the analysis is performed a gene list will be returned. The Pairwise Analysis Results Page lists the genes that are differentially expressed based on the pairwise analysis settings selected. 764 genes passed the filtering criteria –a 1.5 fold or greater change in expression and a raw p-value of at least 0.05 from the t-test. The genes are sorted by fold change and the first 20 genes in the list are displayed.

8. To filter the list using the adjusted p-value (false discovery rate), select “adjusted p” from the pull-down menu and then click the Search button.

9. The list filtered on the adjusted p value contains 624 genes with a false discovery rate less than 5%.

10. To view data and a gene summary for any gene in the list, click the Gene Name.

11. This will bring up a data summary and a One-Click Gene Summary™ (OCGS) for the gene. The One-Click Gene Summary provides a synopsis of current UniGene and Entrez Gene (formerly known as LocusLink) information for the gene.

12. Go back to the gene list by clicking the “Back” button in your browser.

13. Select the Ontology link at the top of the screen to view a summary of the Gene Ontology terms associated with the genes in the list. See the online help system for information about the other reports.

Note: To the view page-specific online help documents for any page, select the question mark icon located in the upper right corner of each page.

14. The Ontology Report lists the Gene Ontology terms associated with the 624 genes in the pairwise results gene list. See the help documents for this page for more information about the Ontology Report.

15. Click on Z-score report in the upper right corner of the Ontology Report window.

16. The z-score report lists the biological process ontologies that are significantly over or under-represented in the gene list (z-score greater than 2 or less than -2, respectively). Select the red arrow in the z-score column (on the right of the screen) to sort the list by z-score for the up-regulated genes.

Z-score reports can be generated for the Molecular Function and Cellular Component ontologies as well.

17. Minimize, move or close the z-score report window. Return back to the main analysis window, and click KEGG. This will bring up a new z-score report for the KEGG pathway terms associated with the differentially expressed genes.

18. In the KEGG column, click on the KEGG icon in the Jak-Stat signaling pathway row to show its KEGG pathway diagram. Differentially regulated genes are highlighted in red.

19. Return back to the main analysis window again and click Scatter Plot.

20. This will bring up a scatter plot of the results. Up-regulated genes are shown in red, and down-regulated genes are green. The gray spots are those that did not pass the analysis parameters. Move the blue box around and click Zoom to see more detail of the scatter plot.

21. Click on data points in the detail in the upper right section of your screen to bring up the gene summary for a specific gene.

Only a few specific aspects of the data set have been explored here. Feel free to examine the data further on your own.