关键词: PCR, GAPDH, 荧光定量

Abstract: Objective In order to find out the best method for GAPDH quantification,the levels of housekeeping gene GAPDH mRNA were detected by Taqman fluorescence quantitative polymerase chain reaction(FQ-PCR),SYBR Green I FQ-PCR and conventional PCR,respectively.Methods GAPDH plasmids were constructed by Blue-White Clone method and checked by PCR and EcoR I.One of the correct clones was selected for isolating a large amount of plasmids.The selected plasmids were then 10-fold diluted and served as standard.Standard curves were prepared using the data from Taqman FQ-PCR,SYBR Green I FQ-PCR and conventional PCR,respectively.The sensitivity and linear range of the three methods were then analyzed.Results The detectable thresholds for Taqman FQ-PCR,SYBR Green I FQ-PCR and conventional PCR were 2.0×10⁴,2.0×10⁷ and 2.0×10⁶ DNA copies,respectively.The linear range for these three methods were 2.0×10⁴～2.0×10¹⁰ copies,2.0×10⁷～2.0×10¹⁰ copies and 2.0×10⁷～2.0×10⁹ DNA copies,respectively.Conclusion Taqman FQ-PCR is a better method for quantifying housekeeping gene GAPDH mRNA than SYBR Green I FQ-PCR and conventional PCR.

Key words: polymerase chain reaction, GAPDH, fluorescence quantification