A sensitive, specific method of real-time quantitative PCR for detection of tissue inhibitor of metalloproteinase-1 gene

Objective To construct the recombinant plasmid and standard curve for detection of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene by real-time quantitative PCR (QPCR) and establish the real-time QPCR assay for accurate detection of TIMP-1.Methods The TIMP-1 gene was acquired by reverse transcription-PCR after isolating mRNA from rat hepatic stellate cells (HSC). The purified product of PCR was subsequently ligased with pGMT-Vector and transferred into Ecoli DH5α. The standard recombinant plasmid was gained from the positive clone.The gradient diluted recombinant plasmid were used as the template to amplify the TIMP-1 gene by two methods, fluorescence QPCR by “Taqman” approach and by SYBR Green I approach.The standard curves made by the two methods were compared and the results of the two methods were compared with the conventional PCR.Results The amplified products of the recombinant plasmid by PCR and gene sequencing confirmed that the pGMT-TIMP-1 gene was successfully cloned. When using the gradient diluted recombinant plasmid as the template to amplify the TIMP-1 gene by the two real-time QPCR,“Taqman” approach can detect 7×10 4～7×10 8 copies , while SYBR Green I approach only detect 7×10 6～7×10 8 copies. The sensitivities of two methods were 7×10 4 copies and 7×10 6 copies, respectively. The melting curve also showed that unspecific amplification appeared when detected by SYBR Green I approach. Meanwhile, both of the two real-time QPCR have the quantitative function compared with the conventional PCR.Conclusion The recombinant plasmid and standard curve to detect the TIMP-1 gene by the “Taqman” approach was good at sensitivity,specificity,quantification and linear function. It can be a standard method of real-time QPCR for detection of TIMP-1.