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 tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) in tissue samples. This method is based on the real-time monitoring of reverse transcriptase-polymerase chain reaction (RT-PCR) with a dual-fluorescence-labeled probe and a sequence detector. A linear correlation existed between assay measurements and input target (r = 0.999). TNF-alpha mRNA was detected in the synovial cells of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and also in the U937, THP1, and HL60 cell lines. Stimulation with interleukin-1alpha (IL-1alpha) caused an immediate increase in the intracellular 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 quantitative detection of TNF-alpha mRNA. The real-time quantitative RT-PCR assay may be valuable for measuring TNF-alpha mRNA expression in clinical samples.
Takahashi
M
M
Department of Molecular Diagnostics and the Department of Rheumatology and Haematology, Tohoku University School of Medicine, Sendai, Japan.
Funato
T
T
Ishii
K K
KK
Kaku
M
M
Sasaki
T
T
D001172:Arthritis, Rheumatoid / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts / Q000737:chemistry*; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D010003:Osteoarthritis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013583:Synovial Membrane / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D005456:Fluorescent Dyes; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha