Establishing a method for detection of human vitamin D receptor using dual real-time fluorescence quantitative PCR / 天津医药

ABSTRACT

Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction (dual real-time 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 puri-fied to construct the VDR/GAPDH recombinant plasmid, which was taken as the standard to analyze the sensitivity and re-peatability 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 amplifica-tion 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.