Using the RefGenes Tool to Find Reference Genes

STEP 1: Choose samples from a biological context similar to those in your qPCR expriment

How to choose the samples you want to analyze?

• Click the New button in the Sample Selection panel. The selection of samples defines which data are used for the analysis.
• Select the organism you’re interested in (in this example: human)
• Select the array type you want to analyze (in this example: human 133_2). For most organisms Genevestigator contains expression data from multiple types of microarrays, e.g. different generations of Affymetrix GeneChips®. On these arrays, genes are sometimes represented by different sets of probes. To keep the analysis results easily interpretable, data from different array types are not mixed.
• Click the Select particular conditions button to select all samples with a certain annotation, e.g. all data from a certain tissue type.
• Select the type of conditions (red) you want to base your selection on (in this example: Anatomy). For each type (anatomy, neoplasms, perturbations, development…) you can browse the corresponding ontologies and select the desired condition(s) (green) (in this example: cardiac muscle).
• Click OK.

Note that you can select multiple tissues. When you select samples for use in the RefGenes tool, you have to focus on microarrays from samples that were collected in conditions similar to those in your qPCR experiment. Don’t make a too general selection, e.g. all human samples: you might end up with genes that are stable in most conditions but not in yours. Don’t make a very specific selection either, e.g. human heart samples from patients taking the same medication as yours. If you want to broaden your study later on with samples from other patients, your reference genes might not be valid anymore. It is recommended to select reference genes in the same organism and the same / a similar tissue type as the one that you used in your experiments.

STEP 2: Select the gene(s) you want to measure in your qPCR experiment

This step is not essential, but it helps you to see whether your target gene(s) is (are) strongly or weakly expressed in the conditions of interest selected in STEP1. This allows you to search for candidate reference genes in a similar range of expression.

How to choose the genes you want to analyze?

• Click the New button in the Gene Selection panel.
• Enter the name of your target gene in the text area (in this example: GOT1) and click OK
• Open the RefGenes tool (if you haven’t done that already). A red box plot representing the distribution of the expression levels of GOT1 in the 68 selected human heart samples appears in the center panel. As you can see, this gene is highly expressed in heart.

STEP 3: Find candidate reference genes

The reference genes that are suggested by GeneVestigator have the following characteristics:

• They have the most stable expression levels across all selected samples (a small boxplot)
• Their overall expression level is similar to that of the target gene(s) of your qPCR experiment

How to find the candidate reference genes?

• Click the Run button in the RefGenes tool. RefGenes will show the top 20 most stable genes with similar expression levels: