Fast and accurate quantitative analysis of cytokine gene expression in human neutrophils by reverse transcription real-time PCR.

In recent years, several studies have brought forward new and exciting discoveries in polymorphonuclear neutrophil research by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the biology of this cell. At present, neutrophils should no longer be regarded as cells that only release preformed mediators, but instead as fundamental contributors for many events that control and regulate inflammatory, immune, and other relevant responses. In this context, a correct methodological analysis of this novel neutrophil function represents a critical step toward a better understanding of how the release of cytokines by neutrophils may influence pathophysiological processes in vivo. We now describe and discuss methods we have recently developed to more rapidly characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool for high-throughput analysis of gene expression in neutrophils.