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GAPDH, a good reference sequence?

Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or, less commonly, as G3PDH) (EC 1.2.1.12) 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 [1] that result in variable expression lev­els across different tissues [2]. 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 [3]. 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 [4].
  • 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 [5] 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 [8] for more comprehensive and detailed normalization and relative quantification strategies.

References

  1. Hara MR, Agrawal N, et al. (2005). S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol, 7(7):665–674.
  2. Radonic A, Thulke S, et al. (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun, 313(4):856–862.
  3. Butterfield DA, Hardas SS, and Lange MB, (2010) Oxidative modified glyceraldehyde-3-phos­phate dehydrogenase (GAPDH) and Alzheimer’s disease: Many pathways to neurodegeneration. J Alzheimers Dis, 20(2):369–393.
  4. Liu Y-J, Zheng D, et al. (2009) Comprehensive analysis of the pseudogenes of glycolytic enzymes in vertebrates: the anomalously high number of GAPDH pseudogenes highlights a recent burst of retrotrans-positional activity. BMC Genomics, 10:480.
  5. Kalyana-Sundaram S, Kumar-Sinha C, et al. (2012) Expressed pseudogenes in the transcriptional landscape of human cancers. Cell, 149(7):1622–1634.
  6. Bustin SA. (2000) Absolute quantification of mRNA using real-time reverse transcription poly­merase chain reaction assays. J Mol Endocrinol, 25:169–193.
  7. Bustin SA. (2000) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 29:23–39.
  8. Bustin SA, Benes V, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4):611–622.

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Additional reading

Sample preparation for successful qPCR—Read about qPCR sample preparation, and what experimental details you should consider for obtaining accurate and consistent results.

Steps for a successful qPCR experiment—Read these recommendations for 5′ nuclease assay design and experimental setup that will help you obtain accurate and consistent results.

Design efficient PCR and qPCR primers and probes using online tools—Simplify planning of your qPCR experiments using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

Author: Rami Zahr, MS, is a Scientific Applications Specialist at IDT.

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