1. Structure and liver cell expression pattern of the HFE gene in the rat
Petra Holmström, Vijole Dzikaite, Rolf Hultcrantz, Öjar Melefors, Kristina Eckes, Per Stål, Nils Kinnman, Bård Smedsrød, Mats Gåfvels, Gösta Eggertsen 
Journal of Hepatology 
Volume 39, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Fig. 1
Schematic drawing of the rat HFE gene with the isolated genomic fragments (A, B and C) arranged in position. Arrows denote primers used for the genomic PCR amplification across the remaining exon-intron boundaries (Pairs 1–5). → (forward primer) and ← (reverse primer).
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
Gene expression profile of the HFE gene in rat using a Multiple Tissue Northern Blot (Clontech, Palo Alto, CA, USA), hybridized with a digoxigenin labeled rat HFE antisense RNA probe, and exposed for 3 h. The HFE transcript was recognized in heart, brain, spleen, lung, liver and kidney, but not in testis or skeletal muscle (upper panel). The strongest response occurred in liver tissue, where a positive signal was detectable after 15 min. As a control, the membrane was probed with a 32P-labeled human β-actin cDNA probe (bottom panel).
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
HFE gene expression in various organs participating in the iron metabolism from normal rat, using Northern blots hybridized with a DIG-labeled HFE specific antisense RNA probe (top panels). Samples consisted of approximately 1.5 μg of poly(A)+-enriched mRNA from each of the investigated tissues or cells. mRNA isolated from rat liver served as positive control. A DIG-labeled rat GAPDH antisense RNA probe was used as loading control (bottom panels). (a) HFE transcript is present in isolated rat hepatocytes. (b) Presence of HFE transcript was found in bone marrow and spleen from normal rat. A faint signal was detected in bone marrow after 30 min exposure. (c) The HFE gene is widely and uniformly expressed in the proximal small intestine of normal rat. HFE mRNA levels were monitored in five individual segments, each 1 cm in length, from the rat proximal small intestine (isolated 1, 3, 6, 8 and 10 cm distal to the gastroduodenal junction).
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
Analysis of the HFE gene expression in isolated fractions of normal rat liver cells. (a) The presence of HFE transcripts as detected by RT-PCR technique. One μg of total RNA was used as a template in each reaction. RNA from rat liver and proximal small intestine served as positive controls for detection of HFE transcripts. RT-PCR analysis were performed on cells from three different isolations for each cell type, using one animal per isolation. (b) Relative quantitation of rat HFE mRNA by singleplex real time PCR analysis. In normal rat, the liver contains 16× as much HFE mRNA as the proximal small intestine, hepatocytes 13.4× as much, Kupffer cells 6.9× as much, and liver endothelial cells 2.5× as much. Calculations were performed according to the ΔΔCT method using eukaryotic 18S rRNA as endogenous control and proximal small intestine as a calibrator. The number of animals in each group was: liver (n=3), proximal small intestine (n=3), hepatocytes (n=4), KC (n=3 of which two were pooled together) and EC (n=1). The range given for the normalized amount of rat HFE mRNA relative to proximal small intestine is determined by evaluating the expression 2 – ΔΔCT with ΔΔCT+s and ΔΔCT−s where s=the standard deviation of the ΔΔCT value. NTC, no template control; EC, endothelial cells; KC, Kupffer cells; and HSC, hepatic stellate cells.
Journal of Hepatology  , DOI: ( /S (03) )