Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method

To investigate cytokine regulation in cells of freshly excised lymphoid tis sues, 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-1 alpha (IL-1 alpha), IL-1 bet a, tumor necrosis factor alpha, (TNF alpha), interferon gamma (IFN gamma), transforming growth factor beta (TGF beta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and residen t peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the fres hly isolated splenocytes and thymocytes. Studies to establish the usefulnes s of these assays for measuring changes in the levels of cytokine mRNA focu sed on IL-1 alpha, IL-1 beta, TNF alpha 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 fres hly isolated cells and from cells following short-term (less than or equal to 26 h) culture. These measurements established basal in vivo levels of sp ecific cytokine mRNAs and demonstrated specific modulation of their levels by cell culture and by the inclusion of immune stimulants (bacterial lipopo lysaccharide or the plant lectin conoanavalin A) in the culture. These data provide new information on both basal and stimulated cytokine levels that is required for valid interpretations of the roles of cytokine expression i n immune regulation.