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Investigation of extrahepatic manifestations in haemochromatosis In severe or advanced haemochromatosis, cardiac and endocrine manifestations may be present

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Using the 2 −ΔΔCT method [ relative expression (fold change) of forkhead box O1 (FoxO1) (5′-AGCTCAAACGCTAGCACCAT-3′ and 5′-GGTGGATACACCAGGGAATG-3′), glucagon (5′-GCCGAGCAAGGCGAGACT-3′and 5′-CATGTCTGCGCCCAAGTTC-3′), insulin (5′-CCTGCCCAGGCTTTTGTCA-3′ and 5′-GGTGCAGCACTGATCCACAATG-3′), X-box binding protein 1 (XBP1) (5′-GGTCTCAGAGGCAGAGTCCAAG-3′ and 5′-AGAGGCAACAGCGTCAGAATCC-3′), Hspa5(BIP) (5′-GAGGACAAGAAGGAGGATG-3′ and 5′-TTGGACGTGAGTTGGTTC-3′), IL6 (5′-GTCAACTCCATCTGCCCTTC-3′ and 5′-TGTGGGTGGTATCCTCTGTG-3′), IL1β (5′-GCCAACAAGTGGTATTCTCCA-3′ and 5′-TGCCGTCTTTCATCACACAG-3′), HIFα (5′-TGGATGGCTTTGTTATGGTG-3′ and 5′-TGGTCACATGGATGGGTAAA-3′), and GAPDH (5′-TTAAGGGCATCCTGGGCTACACT-3′ and 5′-TTACTCCTTGGAGGCCATGTAGG-3′) was normalized to β-actin (5′-GTCGTACCACTGGCATTGTG-3′ and 5′-CTCTCAGCTGTGGTGGTGAA-3′) as the most suitable reference gene (expression level unaffected by the experimental treatment) and relative to the sham group as the calibrator