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 maps 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.