RefGenes - a unique tool to find suitable reference genes
The choice of suitable reference genes is absolutely
crucial in RT-qPCR gene expression analysis. Often, genes from
commercial panels don't work well for one's own biological context.
Ideally, the expression of reference genes should remain unchanged
across samples within the context under study.
RefGenes is an online app from GENEVESTIGATOR that allows users to
search for genes that are most stable across a chosen set of samples
based on microarray data. This set of samples can be chosen
according to experimental conditions or tissue types. For example,
if you are performing a RT-qPCR experiment on mouse liver samples,
you can use RefGenes to identify the set of genes that are most
stable across all microarrays done on mouse liver in GENEVESTIGATOR.
This method offers two major improvements over existing methods
because a) it does not narrow down from a small set of genes (e.g.
commercial housekeeping gene panels), but looks for novel candidates
from a genome-wide set of genes b) it is based on condition-specific
stability. The below schema shows how RefGenes can be used in
combination with existing approaches to yield valuable reference
genes for specific experimental conditions.
Figure 6 of Hruz et al. [PDF]
Our experience with identifying reference genes from
microarray data is to search from a sufficiently large set of
microarrays. We recommend the following:
- At least 50 microarrays
- At least 3 independent studies
- Limit the search to medium or high expression levels
If for a given condition there are not sufficient
microarrays available in the GENEVESTIGATOR database, extend your
selection of microarrays to related or similar conditions. Some
genes in the very low expression range may be proposed by the tool.
Make sure you choose genes that are expressed (non-expressed genes
of course have low variation of expression!), so set the thresholds
for minimum and maximum expression somewhere in the upper ranges of
The RefGenes tool is part of GENEVESTIGATOR and is accessible to users
with a Basic
(free for academics) or Professional
license. Register here.
The GENEVESTIGATOR database, which is queried by RefGenes, contains
data from 14 species, such as human, mouse, rat, Arabidopsis,
Drosophila, yeast, E. coli, and several crop plant species. See the
page for more details.
An article in BMC Genomics has been published about
RefGenes. The reference is:
Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi
L, Verbrugge P, Kalaydjieva L, Bleuler S, Laule O, Descombes P,
Gruissem W and P Zimmermann (2011)
identification of reliable and condition specific reference genes
for RT-qPCR data normalization.
BMC Genomics 2011, 12:156.
Articles citing RefGenes
RefGenes is used across a wide range of biological research labs.
See what the did with this tool by checking articles
in Google Scholar.