RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd).
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.
Harper
Lucy V
LV
Molecular Biosciences, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK. lvharper@dialstart.net
Hilton
Anthony C
AC
Jones
Alan F
AF
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011544:Pseudogenes / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases