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Objective To establish a nested-real-time-PCR for the detection of IL-21R mRNA in peripheral blood mononuclear cells(PBMC)of patients with rheumatoid arthritis,and explore its clinical significance.Methods The outer primers of span intron and inner primers were designed for nested-real-time-PCR.To increase specific template,the concentration of primers and enzyme,cycle times were decreased in the first amplification,and the real-time fluorescent quantity assay was performed in the second amplification.The ?-actin gene was used as internal reference,and the results were presented as the ratios of IL-21R mRNA to ?-actin mRNA.Results IL-21R mRNA was detected in 60 patients with RA and 30 healthy controls.The expression of IL-21R mRNA gene in the patients with RA was increased,and the statistical analysis has significant differences(P0.05),but a significant difference was found in the patients of grade III compared with those of grade I and II(P
RESUMEN
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.