The DHPLC technology can be employed for accurate, absolute quantification of gene expression, which is estimated by the competitive reverse transcription (RT) PCR. Competitive RT-PCR is based on competitive coamplification of a dilution series of known concentrations of internal standard RNA (competitor) together with a constant amount of total RNA (target) in one reaction tube.
The amplified RT-PCR products are verified by restriction fragment analysis. Fragments of expected sizes are then quantified by DHPLC analysis. The use of DHPLC in the quantification process offers a rapid, accurate, and automated measurement of gene expression. It is superior to the time-consuming and labor-intensive gel electrophoresis with the use of radiolabeled or fluorescent components.
In a recent breast cancer study, the quantitative method identified that nine candidate genes were over-expressed in breast tumor cells. The method was also used for studying the differences in quantitative gene expression of α and γ sodium pump subunits of nephron segments from hypertensive rats.