E2 deficiency upregulates IL-7 mRNA and protein concentrations in murine bone marrow cells in vivo. (a) Levels of IL-7 mRNA were analyzed by semiquantitative RT-PCR using whole murine bone marrow isolated from E2-deficient ovx mice and E2-replete mice (sham-operated, and ovx receiving E2 replacement) 2 weeks after surgery (n = 4 mice per group). RNA concentrations were internally controlled using the housekeeping gene GAPDH. IL-7 was evaluated at 30 PCR cycles and GAPDH at 25. IL-7 was confirmed to be below saturation up to 35 cycles and GAPDH up to 30 cycles (data not shown). Data are representative of two independent experiments. (b) Quantitation of IL-7 mRNA. IL-7 mRNA bands in a were quantitated by densitometry using NIH Image and normalized for GAPDH concentrations. (c) IL-7 protein concentrations in the marrow compartments of E2-replete and E2-deficient mice were measured by ELISA in cell-free bone marrow serum immediately following isolation of the marrow, as described in Methods. Data represent average ± SEM of four mice per group. Data points were measured in quadruplicate. *P ≤ 0.05 (ANOVA).