Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress.

The main stage in real-time quantitative PCR is a quantification of gene transcriptomes, in which suitable use of reliable reference genes is critical to normalize accurately. To determine the most stable reference genes in laying hens under heat stress, from a panel of nine typical candidate reference genes, the mRNA transcript of ACTB, HMBS, HPRT1, RPL13, RPL32, 18SrRNA, TBP, TFRC, and YWHAZ was evaluated in the ovary and uterus of both control and heat-stress groups of laying hens. Forty 23-week-old White Leghorn laying hens were housed in two rooms. The control (n = 20) and heat-stress (n = 20) groups were maintained at 21-23 °C and 36-38 °C for 8 weeks respectively. Analysis of this set of genes was done with BestKeeper, geNorm, and NormFinder software programs to find the most stable ones. Candidate reference genes ranked in the uterus of heat-stress and control groups of hens included YWHAZ, HPRT1, HMBS, RPL13, TFRC, ACTB, TBP, RPL32, and 18SrRNA; those in the ovary were YWHAZ, HPRT1, TFRC, HMBS, RPL13, TBP, RPL32, ACTB, and 18SrRNA. The overall results indicated that the most stable genes are YWHAZ, HPRT1, HMBS, RPL13, TFRC, TBP, ACTB, RPL32, and 18SrRNA respectively. In addition, the combination of YWHAZ, HPRT1, and HMBS is suggested as the most stable reference group of genes for more accurate quantitative data normalization in the ovarian and uterine tissues of laying hens under control and heat stress conditions. Lay summary Heat stress influences the expression of many genes in the reproductive tissues of birds. Accurate evaluation of these changes via real-time quantitative PCR depends on the determination of reliable reference genes. In this study, nine candidate housekeeping genes were evaluated, and the most stable were YWHAZ, HPRT1, HMBS, RPL13, TFRC, TBP, ACTB, RPL32, and 18SrRNA.