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).
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
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.
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.