Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RT-PCR technology

The analysis of cytokine profiles plays a central part in the characterizat ion of disease-related inflammatory pathways and the identification of func tional properties of immune cell subpopulations. Because tissue biopsy samp les are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milie u in inflammatory lesions. RT-PCR itself is a qualitative method, indicatin g the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on intern al shortened standards. Recently, online real-time PCR has been introduced (LightCycler(R)), which allows quantitation in less than 30 min. Here, we h ave tested its use for the analysis of cytokine gene expression in differen t experimental in vitro and ex vivo settings. First, we compared quantitati ve competitive RT-PCR with real-time RT-PCR in the quantitation of transcri ption levels of the CD4(+) cell-specific chemoattractant Interleukin-16 dur ing the maturation of monocyte-derived dendritic cells, and found a good co rrelation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoa rthritis as assessed by real-time RT-PCR paralleled differences in the leve l of IL-16 protein in the synovial fluid. Finally, we employed real-time RT -PCR to study the cutaneous expression of several cytokines during experime ntal immunomodulatory therapy of psoriasis by Interleukin-10, and demonstra te that the technique is suitable for pharmacogenomic monitoring. In summar y, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expr ession even with small quantities of tissue. The results obtained do not di ffer from those generated by quantitative competitive RT-PCR. (C) 2000 Else vier Science B.V. All rights reserved.