Detection of APRIL mRNA in peripheral blood of auto-immune diseases using a quantitative real-time PCR method

Objective To establish a SYBR Green Ⅰ quantitative real-time PCR method for detecting the expression of APRIL gene(a proliferation-inducing ligand,APRIL) in peripheral blood of patients with auto-immune diseases,e.g.,systemic lupus erythematosus(SLE) and rheumatoid arthritis(RA),and investigate the relationship of APRIL mRNA expression with pathogenesis and prognosis of auto-immune diseases.Methods Plasmid PGEM-T easy-APRIL was cloned as the standard template.SYBR Green Ⅰ quantitative real-time PCR was set up to examine the expression of APRIL mRNA in peripheral blood of 58 patients with auto-immune diseases and 20 healthy controls using Line Gene FQD-33A Detection System.Results The obtained data were normalized by dividing the copy number of target cDNA by those of GAPDH(glyceraldehycle-3-phosphate dehydrogenase,GAPDH).APRIL expression levels ranges from 3.95 to 192 and mean value was 29.68±4.5.APRIL expression of twenty healthy controls showed range from 3.1 to 18.7 and mean value of 10.56±2.0.APRIL expression levels in patients with auto-immune diseases were higher than those in healthy controls.In auto-immune diseases group APRIL expression levels of untreated patients were higher than those of the other patients,and a statistical significance was found.Conclusions APRIL mRNA was successfully detected by SYBR Green Ⅰ quantitative real-time PCR and the method was accurate and reliable.The expression of APRIL mRNA of auto-immune disease patients was higher than those of healthy controls,and the expression of untreated patients was the highest.This method may be used for further study on the high-level expression of APRIL mRNA in mechanism of auto-immune diseases as well as the development and prognosis of diseases.