Similar sequence-free amplification of human glyceraldehyde-3-phosphate dehydrogenase for real time RT-PCR applications

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme. which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus snaps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies. (c) 2005 Elsevier Ltd. All rights reserved.