Detection of differential display code 3 mRNA by the method of SYBR Green I real time quantitative RT-PCR

Objective To establish the quantitative detection method by SYBR Green I real time quantitative RT PCR. DD3 mRNA copies in the cDNA obtained from different tissues were determined by real-time PCR. Methods The 406bp fragment of DD3 mRNA was amplified from total RNA of LNCaP cells using RT-PCR method, and was ligated by pMD 18-T Simple vector. Combined vectors were transformed into JM109. PCR detecting and sequencing were performed for standard plasmid. Then, we detected the copy concentration of stock solution and standardized serial concentrations by SYBR Green I real time quantitative RT-PCR. Then, the expression level of DD3 mRNAs in normal tissues of breast, bladder, colon, urethra, lung, prostate, seminal vesicle, testis, ovary, as well as prostate cancer were quantitatively determined. Results The 406 bp fragment of DD3 cDNA was successfully cloned into the pMD 18-T simple vector and was verified by sequence analysis. The stable detecting standards were constructed, all samples, except prostate, were negative for DD3 mRNA. The DD3 mRNA expression level had no statistical difference between normal prostatic tissues and BPH. As compared with the normal prostate and BPH tissues, the DD3 mRNA expression level of prostate cancer showed significant statistical difference. Conclusion Quantitative detection of DD3 mRNA by the method of SYBR Green I real time quantitative RT-PCR is stable and reliable. The expression of DD3 mRNA is prostate-specific and has great potential for development of a new molecular diagnostic tool.