Abstract
Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ribosomal RNA (18S rRNA), ribosomal protein L4 (RPL4) and HMBS, RPL4, respectively. BestKeeper analyses showed that 18S rRNA, RPL4 and HPRT1, HMBS were the two most stable reference genes for POT, and POT+N, respectively. Of the ten reference genes, all except B2M showed geNorm M <0.5, suggesting that they were stably expressed in the shell gland tissue. Consensus from these three programs suggested HPRT1 and HMBS could be used as the two most stable reference genes in the present study. Expression analyses of four candidate target genes with the two most and the two least stable genes showed that a combination of stable reference genes leads to more discriminable quantification of expression levels of target genes, while the least stable genes failed to do so. Therefore, HMBS and HPRT1 are recommended as the two most stable reference genes for the normalization of gene expression data at different stages of eggshell formation in brown-egg laying hens. Available statistical programs for reference gene ranking should include more robust analysis capability to analyse the gene expression data generated from factorial design experiments.
Highlights
The chicken reproductive tract is divided into five histologically distinct parts, the ovary, infundibulum, magnum, isthmus, and shell gland
We investigated the stability of ten reference genes in the shell gland region of the oviduct in relation to different stages of eggshell formation and nicarbazin treatment
The data analysed by the three different statistical software indicated that the overall stability of the reference genes was affected by different time-points and nicarbazin treatment, Fig 4
Summary
The chicken reproductive tract is divided into five histologically distinct parts, the ovary, infundibulum, magnum, isthmus, and shell gland (uterus). The shell gland is an expanded pouch-like part of the oviduct where an egg remains for approximately 18–20 hours, during which shell formation takes place [1]. The ovulation occurs 0.5 hour after the preceding oviposition [2]. Reference gene selection for the shell gland of laying hens
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