title: Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes.
abstract: In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.
authors.affiliation: Laboratorio de Biotecnologia Molecular, Departmento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Universidad Andres Bello, Av. Republica 217, 8370146 Santiago, Chile.
authors.forename: Eduardo N
authors.initials: EN
authors.lastname: Fuentes
authors.forename: Diego
authors.initials: D
authors.lastname: Safian
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Valdés
authors.forename: Alfredo
authors.initials: A
authors.lastname: Molina
mesh_terms: D000818:Animals; D005432:Flounder / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D018482:Muscle, Skeletal / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D009752:Nutritional Status; D060888:Real-Time Polymerase Chain Reaction; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000235:genetics
chemical_list: D017526:Receptor, IGF Type 1; D018995:Myosin Heavy Chains