Establishment of the method for detecting human Gfi1 mRNA with SYBR GreenIreal-time fluorescence quantitative ploymerase chain reaction

Objective To establish SYBR GreenⅠreal-time fluorescence quantitative polymerase chain reaction method for detecting human Gfi1 mRNA and studying function of new gene.Methods We designed Gfi1 specific primers and constructed plasmid plox-Gfi1 carring Gfi1cDNA,then purified,quantified spectrophotometrically,calculated copies and used a 10-fold serial dilutions of quantified plasmids.At last,we established standard curve and melting curve to measure Gfi1 using ABI stepone PCR instrument with real-time fluorescent polymerase chain reaction.Results A good linearity was found from 6.36×102 to 6.36×108copies/μl,and the correlation coefficient was 0.996.Melting curve analysis showed a single peak,and Tm was(89.98±0.22)℃.The intraassay and interassay variation of cycle threshold was 1.35%～3.61% and 1.49%～3.42% respectively.Conclusions SYBR Green I real-time fluorescent quantitative polymerase chain reaction of Gfi1 mRNA expression is a rapid,sensitive,specific,repeatable and stable method.It can be used to further research on human Gfi1.