In our experience, real-time PCR of cells assayed both immediately ex vivo and after in vitro culture with activating stimuli has proved useful for determining relative quantity of cytokine mRNA in cells from patients with SLE and control subjects (see Subheadings 4.2. and 4.3.). Parallel studies of intra-cellular cytokine expression confirm the PCR data and provide an indication of the cellular source of the cytokine. We are also measuring mRNA specific for cytokine target genes in peripheral blood cells studied immediately ex vivo to support an important functional role for the cytokine in immune system function. It is this redundant approach using complementary assays that is most likely to generate accurate and interpretable data leading to new understanding and treatments of the altered immune function of patients with autoimmune disease.
This review provides detailed methodology for two currently used approaches for measurement of cytokine mRNA and protein: real-time PCR and intracellular staining using flow cytometry. A more comprehensive list of methods of analysis of cytokine expression is provided, with selected references.
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