Detection of the proliferation-inducing ligand APRIL mRNA in autoim-mune disease using real-time quantitative PCR assay

To establish a SYBR Green 1 real-time quantitative PCR assay to detect the expression of APRIL gene in peripher- al bloods of patients with autoimmune diseases,such as systemic lupus erythematosus(SLE)and rheumatoid arthritis(RA),so as to make certain of the relationship between the expression of APRIL mRNA and the pathogenesis as well as the prognosis of autoimmune diseases,plasmid PGEM-T-easy-APRIL was cloned and was used as the standard quantitative template.The SYBR Green I real-time quantitative PCR assay was set up to detect the expression of APRIL mRNA in peripheral bloods of 58 patients with autoimmune diseases and 20 normal controls using line gene FQD-33A fluorescence quantitative PCR amplifica- tor,and the ratio of APRIL mRNA with that of the reference mRNA of glyceraldehydle-3-phosphate dehydrogenase(GAPDH) was used as the indices of the expression level.It was demonstrated that the expression ranges of APRIL mRNA of 58 patients with autoimmune diseases and 20 normal controls were 3.95～192(mean value:29.68±5.4)and 3.1～18.7(mean value: 10.56±2.0)respectively,indicating a higher expression level of APRIL mRNA in patients with autoimmune diseases than that of normal controls.In addition,the APRIL mRNA expression level of patients without response after long period of treat- ment or with poor response to therapy was even higher than that of patients discovered recently or patients with good response to therapy.However there was no significant difference in the expression levels among these cases.It is evident that the SYBR Green I real-time quantitative PCR assay was successfully established for use to detect the expression of APRIL gene in periph- eral bloods of patients with autoimmune diseases,and this method of assay was proved to be accurate and reliable,thus provi- ding the basis for the studies on the pathogenesis,early diagnosis,prognosis and searching for the novel therapeutic target for autoimmune diseases.