Hydroxymethylbilane synthase mRNA levels are increased in RNA preparations from IRP2^-/- IRP1^+/- IRP2^-/- spleens. Spleen RNA was prepared and Northern blots were probed for levels of hydroxymethylbilane synthase, as described in “Materials and methods.” To evaluate total iron (heme and nonheme) content of tissues in IRP2^-/- mice, we performed ashing and analyzed total iron levels in spleen, brain, and liver (Table 4). Total spleen iron was unchanged in IRP2^-/- and IRP1^+/- IRP2^-/- mice relative to WT controls. Total brain iron was also unchanged, while total liver iron was increased in IRP1^+/- IRP2^-/- mice relative to wild type. Kb indicates kilobase.