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

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.