What internal controls are you using? Housekeeping genes are frequently used as internal controls. However, they may not be the best choices due to lesser known functions and the presence of pseudogenes, some of which, as in the case of GAPDH—used here as an example—may be expressed.
Jul 5, 2013
Revised/updated Aug 22, 2016
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or, less commonly, as G3PDH) (EC 184.108.40.206) is an enzyme of ~37 kDa that plays an important role in glycolysis. GAPDH is a popular housekeeping standard used in gene expression studies. Many researchers are not aware, however, of the difficulty of using this mRNA as a reference in qPCR assays:
- In addition to its activity in the glycolytic pathway, GAPDH plays other roles in the cell  that result in variable expression levels across different tissues . Studies have shown diverse functions and activity of this protein from DNA and RNA binding to key roles in neurodegenerative disease such as Alzheimer’s . Therefore, the variable expression in these different cell types may result in GAPDH serving as a poor reference gene.
- The genomes of the most popular model organisms contain many GAPDH pseudogenes—60 in human, 285 in mouse, and 329 in rat .
- Some GAPDH pseudogenes are expressed. These pseudogenes have identical or nearly identical sequences to the active, target GAPDH transcript, and therefore primers or probes spanning exon junctions will detect the presence of the pseudogenes along with the cDNA of the active transcript.
- For samples treated with DNase, it is also possible to retain some of the genomic DNA in which the pseudogenes reside, again resulting in their unintended detection, which will contribute, at least fractionally, to the signal of the assay targeting expressed GAPDH.
Use of additional reference genes
These factors can complicate interpretation of experimental results  and will impact the relative quantification of other assays in the experiment. GAPDH is not the only common reference gene that merits such scrutiny; it is likely that many reference genes also have variable expression across some cell types and disease states. However, use of multiple reference genes can reduce the quantification error introduced by the variability caused by a single reference gene.
Other normalization options
An alternative approach is to normalize against total cellular RNA content (molecules/g total RNA and concentration/g total RNA) [6,7]. Researchers who include multiple reference genes will benefit from normalization to RNA concentration as they will be able to identify reference genes with variable expression and remove them from the experiment. IDT also recommends referring to the MIQE guidelines  for more comprehensive and detailed normalization and relative quantification strategies.