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 characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples 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 milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating 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 internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.
Blaschke
V
V
Department of Dermatology, von-Siebold-Str. 3, D-37075, Goettingen, Germany. vblasch@gwdg.de
Reich
K
K
Blaschke
S
S
Zipprich
S
S
Neumann
C
C
D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000627:therapeutic use; D019410:Interleukin-16 / Q000096:biosynthesis* / Q000235:genetics; D016209:Interleukin-8 / Q000378:metabolism; D009000:Monocytes / Q000166:cytology; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism; D011565:Psoriasis / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D023063:Receptors, Interleukin-8B / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D019410:Interleukin-16; D016209:Interleukin-8; D012333:RNA, Messenger; D023063:Receptors, Interleukin-8B; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma