Transcriptome analysis of orofacial development in mouse embryo

The following tutorial walks through the identification of biological themes associated with gene clusters identified in a murine orofacial development time series.

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

2. Click on the magnifying glass icon next to the “Timecourse::Filter I::Filter II” project, which contains data for all genes on GeneChip® U74Av2 Mouse Genome array. The filtered sub-project contains 411 genes that were differentially regulated with at least a 1.5 fold change across the time series and statistically significant (5% false discovery rate based on ANOVA followed by correction for multiple testing using the method of Benjaminiand Hochberg).

3. At the top of this page is a Project Summary of the project data, listing the time points examined in this project. The orofacial development time series examines gene expression in mouse orofacialtissue at 12, 13 and 14 days of gestation.

4. Select Cluster from the list of analysis options. This can be found in the second gray bar marked "Project Analysis: Filter II" on the right side of the bar.

5. Partition clustering will be used to separate the 411 genes into groups based on expression pattern. Set the "Clusters" in the PAM section to 2 from the pull-down menu, and use the defaults for the other settings. Click the "Search" button. A graph showing the center for each cluster will be displayed.

6. Each line graph summarizes the gene expression pattern for that cluster (each gene in a cluster has an expression pattern more similar to the pattern shown in the graph than to the pattern associated with each of the other clusters). The number of genes in each cluster is listed below the graphs.

7. Silhouette widths measure how well the genes in each group are clustered and can be used to select the best number of clusters for a set of genes. See the help documents for this page for more information about silhouettes.

Note: To view page-specific help documents for this or any page, select the question mark icon located at the upper right page corner. On this page, help documents are available for PAM and silhouettes (including the interpretation of the mean silhouette width).

8. To view the list of genes associated with a cluster, click on the graph. For this tutorial, select the graph for the cluster with 269 genes showing up-regulation during the time series.

9. The resulting heat map shows the expression pattern of the 269 genes in the cluster. To find out more about any of the genes in the list, select the gene name to access the One-Click Gene Summary™ for that gene.

10. The One-Click Gene Summary is a synopsis of current UniGene and Entrez Gene (formerly LocusLink) annotation for the gene.

11. To get an overview of the gene families that have been differentially regulated at this time point, click on the Ontology link near the top of the screen to bring up the Ontology Report.

12. The Ontology Report lists the GO terms of genes in the previous gene list. Currently, they are displayed heirarchically. Click on Z-score Report to show only the significant terms.

13. The z-score report lists the biological process ontologies that are significantly over or under represented in the cluster. Click on Molecular Function to view the z-score report for the molecular function ontologies.

Z-score reports can be generated for each of the clusters. The biological themes associated with each cluster are very different.

14. The last part of this tutorial walks through the use of the “Search by Gene Ontology” feature to isolate a sub set of genes based on their biological function. This feature will be used to identify a sub set of genes with the molecular function ontology “transcription factor activity”.

15. Go back to the gene list page that you opened earlier (it lists 269 genes and has a heat map). Select the Analysis link in the upper right of the page.

16. Select Gene Function on the gray bar in the middle of your screen marked "Project Analysis: Filter II."

17. Select Molecular Function just below the gray bar on the right side of your screen.

18. Select “transcription factor activity” from the pull-down menu marked "Ontology" and then click on the Search button.

19. There are 27 genes with the molecular function ontology “transcription factor activity” that are differentially expressed during the period of orofacial development examined here.