Reference Gene Selection in Adipose and Muscle Tissues of Fat-tailed Lori-Bakhtiari Lambs

Abstract

BACKGROUND: Fat-tailed sheep breeds have a unique ability to tolerate periods of negative energy balance due to seasonal changes in feed availability. This ability is attributed to presence of fat-tail as a body energy reserve, however the exact underlying mechanisms controlling the response of adipose tissue depots to variations in energy balance in fat-tailed breeds are not well understood. OBJECTIVES: As definition of a set of stable reference gene is an absolute prerequisite of any gene expression study, therefore the current research was conducted to define the most stable reference genes in adipose tissue depots and muscle of fat-tailed Lori-Bakhtiari lambs during periods of negative and positive energy balances. METHODS: Eighteen fat-tailed Lori-Bakhtiari male lambs were divided into 3 groups according to their body- weight. The experiment was consisted of an adaptation period (2 weeks), negative energy balance period (3 weeks), followed by positive energy balance period (3 weeks). The 3 groups of lambs were randomly selected and slaughtered at the beginning and end of negative energy balance and at the end of positive energy balance to collect samples of muscle and adipose tissue depots. RESULTS: The stability of the reference genes differed among different tissues and also between various depots of adipose tissue. Average of ranking by different software programs showed that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), B-actin and peptidylprolyl isomerase A (PPIA) were the 3 most stable reference genes in mesenteric adipose tissue, whereas in fat-tail adipose tissue, PPIA, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and RNA polymerase II subunit A (POLR2A) were considered as genes with least expression variability during periods of negative and positive energy balance. B-actin, YWHAZ and phosphoglycerate kinase 1 (PGK1) were defined as the most stable reference genes in longissimus dorsi muscle tissue of Lori-Bakhtiari lambs. CONCLUSIONS: The results of the current study demonstrate that the stability of the reference genes varied between mesenteric and fat-tail adipose tissues and the level of energy balance affects the stability of the refer- ence genes. In addition, ranking of the reference genes differs among different software programs possibly due to different mathematical algorithms used by different programs.