Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were developed for the quantification of expression of the genes for human interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-15, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β and for the endogenous reference hydroxymethylbilane synthase (HMBS). The assays detected as little as five plasmid copies and were 100% specific. The creation and integration of a calibration sample into the assays permitted their calibration across experiments. To handle the high number of generated data, the correlator of advanced real-time assays (CARTA) software was designed to organize samples and to automatically control and analyze TaqMan™ real-time RT-PCR data. The RT-PCR assays were applied to quantify levels of cytokine gene expression in human palatine tonsils at excision and during 4 days of histoculture. Similar longitudinal patterns of cytokine gene expression were observed in all donors, but the variations in spontaneous expression levels between donors were large. The expression levels in histocultures were constant over time and similar to the expression levels at excision except for IL-6 and IL-8, which markedly increased following the first 24 h of culture, possibly due to the initial stress. The standardized and calibrated RT-PCR assays quantify gene expression of human cytokines proved sensitive and specific for the investigation of cell behavior at the molecular level and the newly established CARTA software, a reliable tool for rapid data handling. Tonsil histocultures could serve as a valuable ex vivo model system for further, donor-dependent, studies on activation or repression of cytokine gene expression.