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.
Molecular Biosciences, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK. lvharper@dialstart.net
Lucy V
LV
Harper
Anthony C
AC
Hilton
Alan F
AF
Jones
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