Nested real-time PCR for detection of human FOXP3 mRNA in peripheral blood mononuclear cells / 临床检验杂志

ABSTRACT

Objective To develop a nested-real-time PCR method for the detection of FOXP3 mRNA in human peripheral blood mononuclear cells(PBMC).Methods The outer primers were designed by span intron,and the inner primers and TaqMan probe were designed according to the amplified sequence.During the nested real-time PCR,decrease primers concentration and cycle times were decreased in first PCR amplification to increase specific templates,then quantitative real-time fluorescence was detected in the second PCR amplification.The standard curve was created by FOXP3 and beta-actin recombined plasmid DNA respectively,and the results were presented as the ratios of FOXP3 mRNA to beta-actin mRNA.Results Total mean Ct(threshold cycle) in 6 tests was 14.66.The mean CV(coefficient of variation) was 5.81%.The standard curve showed a fine linear relationship between Ct and template concentration,and the correlation coefficient was 0.999.The sensitivity was 100 times higher than normal real-time PCR by reach 100 copy.Conclusions Nested-real-time-PCR for the detection of FOXP3 mRNA in human PBMC is a simple,repeatable,high specific and sensitive method to evaluate immune function and observe therapeutic effects in certain diseases for clinical routine application.