Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status

This study was designed to investigate the stability of 10 candidate reference genes, namely ACTB, B2M, GAPDH, HMBS, LBR, POLR2B, RN18S, RPS17, TBP, and YWHAZ for the normalization of gene expression data obtained by quantitative real-time polymerase chain reaction (qPCR) in studies related to feed intake of chicken. Samples were isolated from hypothalamus under three different nutritional status (ad libitum, fasted for 24 hr, fasted for 24 hr then refed for 2 hr). Five different algorithms were applied for the analysis of reference gene stability: BestKeeper, geNorm, NormFinder, the comparative Delta Ct method, and a novel approach using multivariate linear mixed-effects modelling for stable reference gene selection. TBP and POLR2B were identified as the two most suitable and B2M and RN18S as the two least stable reference genes for normalization. Despite our review, the current literature showing that RN18S is one of the most commonly used reference gene in chicken gene expression studies, its applicability for normalization should be evaluated before each qPCR experiment.