Abstract
To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10(-18) mol) amounts of the mRNA for seven cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), transforming growth factor beta (TGFbeta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1alpha, IL-1beta, TNFalpha and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (