Detection of TGF-βRII mRNA in peripheral blood mononuclear cells from rheumatoid arthritis patients using nested-real-time fluorescence quantitative PCR

Objective To develop a nested-real-time fluorescence quantitative PCR method for detection of TGF-βRII mRNA in human peripheral blood mononuclear cells(PBMC),and analyze the clinical implications of TGF-β RII in RA patients.Method The outer primers of span intron and inner primers were designed for nested-real-time fluorescence quantitative PCR.To increase specific template,the concentration of primers and enzyme,cycle times in the first amplification were decreased,and the real-time fluorescent quantity assay was performed in the second amplification.The β-actin gene was used as internal reference,and the results were presented as the ratios of TGF-β RII mRNA to β-actin mRNA.Results The standard curve showed a fine linear relationship between Ct(cycle threshold)and logarithm of template concentration,and the correlation coefficient was 0.999.The sensitivity reached 101 copies.The TGF-β RII gene expression in PBMC of RA patient was increased compared to healthy controls[(0.45±0.11)vs(0.37±0.10),P0.01].The ratio of TGF-βRII to β-actin in PBMC of RA patients before treatment was elevated compared to that after treatment[(0.46±0.11)vs(0.40±0.11),P0.01].Conclusions Nested-real-time fluorescence quantitative PCR is a simple,repeatable,high specific and sensitive method for the detection of TGF-β RII mRNA in human PBMC.The expression level of TGF-β RII mRNA may be helpful for diagnosis of RA and evaluation of therapeutic effects.