RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd)

Mol Cell Probes. 2003 Oct;17(5):261-5. doi: 10.1016/s0890-8508(03)00063-x.

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript.

MeSH terms

  • Base Sequence
  • DNA Primers / chemistry*
  • DNA Primers / genetics
  • Gene Expression / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics*
  • Humans
  • Molecular Sequence Data
  • Pseudogenes / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • DNA Primers
  • Glyceraldehyde-3-Phosphate Dehydrogenases