A Sensitive and Specific Method of Real-time Fluorescence Quantitative Polymerase Chain Reaction Using SYBR GreenI for Detection of c-myc Gene

Objective To construct the recombinant plasmid and standard curve for detection of c-myc gene by real-time quantitative PCR(FQ-PCR) using SYBR GreenⅠ and establish the FQ-PCR assay for accurate detection of c-myc gene. Methods The c-myc cDNA was acquired by reverse transcriptase polymerase chain reaction(RT-PCR) after isolating total RNA from tissue of human esophageal squamous cell carcinoma(ESCC). The purified product of PCR was subsequently ligated with pGEM-T Easy vector and transferred into bacterium DH-5α. The recombinant plasmid picked out from positive clones was amplified by PCR,digested with restriction endonuclear EcoRⅠ and sequenced. The mass concentration of the recombinant plasmid was measured and transformed to copy concentration. Then the recombinant plasmid was diluted to series standard concentration and amplified by FQ-PCR. Results That c-myc recombined with pGEM-T Easy vector was proved by digestion and PCR amplification and sequence analysis. The standard curve for detection of c-myc gene was constructed with good correlation with Ct(cycle threshold),the correlation coefficient(r2) ranged from -0.99 to -1.00,the slope ranged from -3.1 to -3.6. Conclusion The recombinant plasmid and standard curve to detect the c-myc gene by SYBR GreenⅠ approach was good in sensitivity,specificity and linear function. It can be used as a standard method of FQ-PCR for detection of c-myc gene.