EXAMPLE

Identifying reference genes for mouse liver

Preliminary remark: the below example was carried out with RefGenes and was experimentally validated. The results are published in the RefGenes article ([PDF]).

Many laboratories work with liver from mouse or other organisms. Due to its particular organ function, the expression of commonly used reference genes may often be unstable in this tissue type. To identify novel candidate reference genes for mouse liver, we chose to work with the mouse Affymetrix 40k array, as there are many liver samples for this platform in GENEVESTIGATOR. We therefore selected all mouse liver samples using GENEVESTIGATOR's data filtering options:
  • Add new array selection
  • Select organism (in this case: mouse)
  • Select platform (in this case: Affymetrix mouse 40k array)
  • Select by annotations (under "Anatomy", select "Liver").
This resulted in a set of 197 microarrays (in the meantime, there are more than 600 mouse liver samples).

In this example, we did not have a particular target gene of interest to be amplified by RT-qPCR, as we aimed at comparing the stability of novel versus commonly used reference genes. We therefore created a gene selection with four reference genes that had previously often been used in mouse liver (GAPDH, TUBB, ACTB, and HPRT). These four genes are displayed in the top section of the RefGenes results graph (see screenshot below).

screenshot example 1

In a second step, we set the range of expression for candidate reference genes to be in a similar range as these four genes (white area in above graph).

Finally, we triggered the search for genes that are stable in mouse liver and RefGenes displayed the top 25 most stable probe sets. We selected four probe sets representative for different expression ranges and designed primers for the corresponding genes (see Hruz et al., 2011, for more details).

In order to validate the stability of expression of these four novel reference gene candidates, an RT-qPCR experiment on 16 mouse liver samples was performed. The data were processed with geNorm, with a sequential leave-one-out procedure. The results clearly show that the candidates proposed by RefGenes were more stable than the four commonly used reference genes GAPDH, TUBB, ACTB, and HPRT.

screenshot example 1


Further examples and validation experiments are shown in the RefGenes publication. In particular, we extrapolated from mouse microarray data to find reference genes for liver from other mammalian species.