Finding Candidate Reference Genes in the Commercial Version of Genevestigator

We will do the same exercise as above in the commercial version of Genevestigator. The difference between the free and commercial version of RefGenes is the number of target genes you can select. In the free version you have to select one gene and then gradually add all other genes one at a time. The commercial version allows you to load as many target genes as you want simultaneously. As a consequence, you can select multiple probe sets for the same gene. All VIB scientists have free access to the commercial version of Genevestigator via their VIB email address. If you don’t know your VIB email address, check the Who’s Who of VIB.

• Open a browser and go to the Genevestigator website
• If it’s your first time to access Genevestigator, create an account by clicking join now button. You will be redirected to a new window in which you will give some personal information including a valid VIB email address. Click Register and check your email to activate your new account. Go back to the GeneVestigator website
• Choose the research field you want to investigate: pharma/biomediacal or plant biology by clicking the corresponding button
• Click Start
• Use your VIB email address and password to login to Genevestigator.
• This will automatically open a Genevestigator startup page in your browser. Keep this page open during the analysis. Closing this page will close Genevestigator.
• Genevestigator is opened automatically

Open the RefGenes tool by clicking its icon in the Further tools section and select the liver samples of the mouse 430_2 arrays as explained in the previous exercise.

Check the expression stability of the 4 commonly used reference genes

• Click the New button in the Gene Selection panel to create a new selection. The selection of samples defines which data are used for the analysis.
• Enter the names of the 4 commercial reference genes in the text area and click OK

I still remove probe sets with an _s or _x since they do not specifically bind to one single gene: Finally you end up with an expandable list of the genes you asked for and you can tick or untick them to control the display of their expression data in the main window. By default all probe sets are ticked so you can see how stable the commonly used reference genes are expressed in the 651 mouse liver samples that are stored in Genevestigator: As you can see, the expression levels of the commonly used reference genes in the selected mouse liver samples is pretty variable which is also confirmed by their relatively high SD values.

The next step of selecting the 4 most stable candidate reference genes with medium expression levels is exactly the same as described above for the free version of RefGenes.

Create a new gene selection with 20 found candidate reference genes and call it mouse_references

• Click the New button at the top of the main window to create a new selection.
• To change the name of the selection right click the name in the Gene selection panel and select Rename

Identify perturbations where the mouse_references genes show more than 1,5 fold differential expression using the Condition perturbations tool

• Click the Home button at the top to go back to the tools overview page.
• Click the Perturbations tool in the Condition Search tools section
• Make a New Sample selection including all mouse 430_2 arrays. Untick all genes except for the first one and filter the long heatmap for at least 1.5 fold change differential expression:

You now get a list of mouse samples in which the gene is not stably expressed so you can check if any of these samples is related to the samples in your study. Hover your mouse over the name of a sample to see more details about the sample. You can do this for each of the candidate reference genes and select the ones that best fit your needs

Exercise on selecting reference genes for metacaspases in Arabidopsis thaliana.

In a geNorm pilot experiment you analyze a set of candidate reference genes in a representative set of samples that you want to test in your final experiment. Based on the M-values and CVs that are calculated by qbase+, you can choose the genes that most satisfy the criteria for a good reference gene.