Quantification of human EZH2 by using SYBR green I real-time fluorescent polymerase chain reaction

Objective:To establish a real-time fluorescent polymerase chain reaction method for quantifying human EZH2.Method:The EZH2 fragment in pure form from classical RT-PCR was cloned to pGEM-T vector, and recombinant plasmid pGEM-EZH2 was purified and quantified spectrophotometrically.Standard curve was established using a series dilution of quantified plasmids to measure EZH2 using SYBR Green I real-time fluorescent polymerase chain reaction and the characteristic of specific EZH2 amplicon was analysed by melting curve.Result:The method can detect as low as 10 copies. A good linearity was found from 101 to 108 copies/reaction and the correlation coeffecient was -1.0.The intraassay and interassay variation of 104 copies/reaction was 1.0% and 2.7%, respectively. Melting curve analysis showed a single peak, and Tm was(83.42±0.13)℃.Conclusion:SYBR Green I real-time fluorescent quantitative polymerase chain reaction which is specific, sensitive and accurate can be used to further research on human EZH2.