Objective:To study the effects of different primers and data analysis methods on quantitative real-time PCR. Methods: Four pairs of different primers were designed for target genes CCND1 and C-JUN using Primer Express 2.0 software. These primers were then used for quantitative PCR amplification and the differences in expression levels between paclitaxel sensitive and resistant lung cancer cell lines were calculated using 2(-DeltaDeltaC(T))method,Pfaffl method and the relative standard curve method,respectively. Results:The expression levels obtained from different primers were significantly different (P < 0.05). In the three kinds of calculation methods,Pfaffl method has no significant difference with the relative standard curve method (P > 0.05),however,2(-DeltaDeltaC(T))method has significant differences with other two kinds of methods (P < 0.05). Conclusion:The amplification efficiency of primers have significant impact on quantitative PCR analysis. The Pfaffl analysis method in the evaluation of relative real-time PCR may be more accurate.