Genevestigator exercise on Liver-specific genes in human

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Goals

• Learn to create gene selections
• Learn to interpret results from meta-profile analysis
• Learn basic workflow with the biomarker search tool
• Learn basic workflow with the clustering analysis tool

Context of the exercise

You will use the meta-profile analysis tool to study a gene specifically expressed in the liver: “Cytochrome P450 family 2 subfamily A, polypeptide 6” The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. You will use Genevestigator to gather more information on this gene.

Start the application.

Follow the instructor’s indications to log on to Genevestigator.

Choose the organism and the genes you wish to analyze

• Choose to work with human data:

Click on the (+) button above the array selection panel and create a new selection Choose “Homo Sapiens” Choose “U133_2: Human Genome 47 Array” Select “High quality arrays only” Check the box next to “Select All” Label this selection as “human_47k_all_arrays”

• Enter your genes of interest:

Click on the (+) button above the gene selection panel and create a new selection Choose “Homo Sapiens” Choose array type as above Enter the following probeset 1494_f_at: Cytochrome P450 family 2 subfamily A, polypeptide 6 Label this selection: “Cyt P450”

View and interpret the results you obtain for this gene

• Click on the “Scanner” Tab in the results panel, set the maximum range to 150’000: What do you see?

Use the magnifying glass to find the array on which the signal intensity is higher than 130’000 To which experiment does this array belong? Which part of the anatomy and genetic background were studied on that array? Give the exact signal intensity measured.

• Click on the “Samples” Tab. What kind of information can you obtain from this plot?

Find the HCC_arrays (within the first quarter of the list). Can you identify which 2 anatomical categories were studied in this experiment? Was the gene present or absent on these arrays? Do you see a strong difference between the 2 anatomical part studied?

• Click on the “Anatomy” Tab. How are both genes expressed in different tissues

Choose the “linear” signals Name the 3 anatomical parts in which the gene is the most expressed. What are the signal intensities in these parts?

• Click on the “Neoplasms” Tab. What can you observe?

Go to the liver neoplasms. Name the liver neoplasm type for which the gene has the strongest signal intensity. Find 3 other cancer types in different organs than the liver in which the gene is highly expressed. What are the signal intensities and the number of arrays in each of these cancer types?

• Click on the “Conditions” Tab. What can you observe?

Which are the 3 experiments in which the gene is the most upregulated? Give the fold change of the gene in these experiments. Among these 3 experiments there is one cancer study. On which type of cancers were the measurements made? Looking back in the neoplasm tool. Can you find some cancer data that validates the observation made in the conditions tool?

• Click on the “Genotype” tab. What can you observe?

In which genetic background is CytP450 the most upregulated? What is the fold change in that experiment?

Biomarker search

The meta-profile analysis of Cytochrome P450 family 2 subfamily A, polypeptide 6 has shown that this gene is:

• • Upregulated in “Mucociliary differentiation (late)”
  • Upregulated in “breast cancer” study 1
  • Upregulated in Mucociliary differentiation (intermediate)”

You will now search for biomarkers which have similar properties. Click on the “Biomarker search tool”, select the “Conditions Tab”, and set the “limit list” to 50 genes. Select the similar properties by ticking the appropriate boxes. Select all genes as base. Run the biomarker search. How are these biomarkers regulated in “anaplastic large cell lymphoma” studies? Save the list of biomarker by clicking on the “copy” icon next to the “run” icon, and by pasting the list as a new gene selection. Call that selection: “Similar to CytP450”

Clustering analysis

• Biclustering of downregulated genes according to “conditions”

Select the “Similar to CytP450” list and click on the clustering analysis tool Choose the biclustering tool Choose “Conditions” for the Select Profile Choose “downregulated” for the Bicluster Run the clustering, and view the various clusters available (flip between clusters at the right of the window) Select bicluster n°:15. In which types of cancer are the genes within this cluster downregulated? How is CytP450 regulated in the similar cancer experiments? To verify this, select CytP450 in your gene selection window, go to the metaprofile analysis tool, and click on the condition tab. To facilitate the visualization set the “View conditions” menu as “tree”. All the conditions will be ordered by families. Look for the appropriate cancer experiments. How is CytP450 regulated in these cancer types?

• Hierarchical clustering according to the anatomy

Select the “Similar to CytP450” list and click on the clustering analysis tool Choose the Hierarchical Clustering tool Choose “Anatomy” for the Select Profile, keep the leaf ordering to none Choose “Cluster genes” and run clustering Choose “Cluster profiles” and run clustering A cluster of 6 probesets is expressed in hepatocytes and the liver. Using the tooltip functions, can you tell how many genes are actually present in that cluster? Give the name of these 2 genes. Select this cluster by clicking on the appropriate node and copy it by clicking on the “copy” button next to the “run” button on the top of the window. Open a new gene selection window and paste your selection by clicking on Ctrl V. Name that selection “Cluster 1” and click on OK. You have now saved the list of probesets from that cluster. View the expression profiles of these 2 genes in the anatomy? Are “Cytochrome P450 family 2 subfamily A, polypeptide 6” and “Cytochrome P450 family 2 subfamily A, polypeptide 7” expressed in similar anatomical categories?

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