Measurement of tumor necrosis factor-alpha messenger RNA in synovial fibroblasts by real-time quantitative reverse transcriptase-polymerase chain reaction

We applied a real-time quantitative assay to determine the expression of tu mor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in tissue sample s. This method is based on the real-time monitoring of reverse transcriptas e-polymerase chain reaction (RT-PCR) with a dual-fluorescence-labeled probe and a sequence detector. A linear correlation existed between assay measur ements and input target (r = 0.999). TNF-alpha mRNA was detected in the syn ovial cells of patients with rheumatoid arthritis (RA) and osteoarthritis ( OA) and also in the U937, THP1, and HL60 cell lines. Stimulation with inter leukin-1 alpha (IL-1 alpha) caused an immediate increase in the intracellul ar level of TNF-alpha mRNA. In particular, the relative copy number of TNF- alpha mRNA increased dramatically from 15 to 3554 in synovial cells from RA patients. This method is simple, accurate, and sensitive for the quantitat ive detection of TNF-alpha mRNA. The real-time quantitative RT-PCR assay ma y be valuable for measuring TNF-alpha mRNA expression in clinical samples.