EXPRESSION ANALYSIS OF THE EQUINE HEPCIDIN GENE BY qPCR
José P. Oliveira Filho1, Peres R. Badial1, Paulo H.J. Cunha1, Taís F. Cruz2, João P.
Araújo Jr2, Thomas J. Divers3, Nena J. Winand4, Alexandre S. Borges1.
Introduction - Hepcidin is an important peptide for systemic iron homeostasis. Hepcidin
up-regulation is particularly useful during acute inflammation, restricting iron availability
necessary for pathogenic microorganism growth, before adaptive immunity occurs.
Objectives - We recently cloned and sequenced equine mRNA hepcidin. Now we
propose to verify equine hepcidin expression in different tissues collected from four
healthy horses in a commercial abattoir. Methods - Total RNA was extracted using
Trizol®, treated with DNase and cDNA synthesized by ImProm-IITM RT System.
Quantitative PCR (AB7300 Real-Time PCR Systems with Power SYBR® Green) was
applied for equine hepcidin and normalized with Equus caballus •-actin mRNA. A
standard curve based method for relative real time PCR data processing was used. For
each animal the basal expression of liver hepcidin was considered 1.0 and all other tissues
were calculated proportionately. Tukey Test was performed to determine whether
expression hepcidin were different among tissues. Results - The relative concentration of
mRNA was 1.0 liver, 8.42x10-4 cervical spinal cord, 5.18x10-4 cerebral cortex, 1.2x10-4
lung, 3.1x10-5 duodenum, 2.5x10-5 stomach, 1.3x10-5 spleen, 9.0x10-6 kidney, 3.0x10-6
skeletal muscle, and 2.5x10-6 bladder. Discussion and Conclusions -Expression of
equine hepcidin was highest in the liver. Although the expression in other tissues was
low, hepcidin expression in cervical spinal cord was significantly higher (p < 0.001) than
in other tissues. These results of hepcidin expression in equine tissues will be helpful for
additional studies on hepcidin and iron metabolism. The procedures described were
previously approved by Institutional Animal Care and Use Committee (109/2007).
1
Department of Veterinary Clinical Science, São Paulo State University – Unesp – FMVZ – Brazil.
Department of Immunology and Microbiology, Sao Paulo State University – Unesp – FMVZ – Brazil.
3
Department of Clinical Science, Cornell University – Ithaca – N.Y. – USA.
4
Department of Molecular Medicine, Cornell University – Ithaca – N.Y. – USA.
E-mail: [email protected]
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP
(07/07344-6, 07/05008-9).
2