Hematological indices and hepcidin mRNA levels in phlebotomized mice. (a) Hematological parameters: RBC (10⁶/ml), HGB concentration (g/dl), HCT (%), and MCV (femtoliter [fl]). Results are expressed as mean ± SD for n animals. (b) Hepcidin mRNA content in liver of control and phlebotomized mice as determined by Northern blot analysis. Twenty micrograms of total liver RNAs were electrophoresed, blotted, and hybridized with both hepcidin and 18S-labeled probes (as described in Methods). Autoradiography of a typical experiment. (c) Relative changes in hepcidin mRNA level (18S normalized, arbitrary units) as calculated using a STORM850 PhosphoImager and ImageQuant 5.0. Results are expressed as mean ± SD for n animals, and statistical analysis was performed using the Student t test (unpaired, two-tailed). *P < 0.001. (d) Level of specific hepc1 transcripts in the liver as measured by RT-PCR (as described in Methods). Following PCR, the amplified products (171 bp for hepc1 and 250 bp for β-actin) were separated by electrophoresis on 1.5% agarose gel.