Abstract: Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction (dual realtime PCR) assay to detect human vitamin D receptor (VDR) and glyceraldehyde- 3- phosphate dehydrogenase (GAPDH). Methods GAPDH gene was used as the internal control. The specific primers and TaqMan probes were designed by Primer Premier 5.0 software, which were applied to detect the VDR/GAPDH mRNA levels. The obtained PCR products were purified to construct the VDR/GAPDH recombinant plasmid, which was taken as the standard to analyze the sensitivity and repeatability of the method. Results The amplification products were confirmed as the specific fragment of VDR/GAPDH by DNA sequencing instrument. The results showed that the sensitivity, linear range, the determinate coefficient, the amplification efficiency, the intra-assay and inter-assay coefficient of variation were 40 copies/μL, 4.00×101-4.00×105 copies/μL, 0.998, 96.10%, 0.09%-1.21%, 0.17%-0.51% for VDR, and 40 copies/μL, 4.00×101-4.00×105 copies/μL, 0.999, 85.15%, 0.35%-0.88%, 0.51%-2.46% for GAPDH, respectively. Conclusion These results demonstrate that the dual real-time PCR assay with high sensitivity and specificity can detect the relative expressions of human VDR by single reaction tube, which can effectively shorten the time and reduce the experimental error.

Key words: receptors, calcitriol, polymerase chain reaction, glyceraldehyde- 3-phosphate dehydrogenases, vitamin D receptor, dual real-time fluorescence quantitative PCR