Identification of reliable reference genes for quantitative real-time PCR in lung and heart of pulmonary hypertensive chickens

Accurate normalization in real-time quantitative PCR is an important step in quantification of gene transcription pattern, in which proper application of stable reference gene(s) is crucial. To identify the most stable reference gene (s) in pulmonary hypertensive chickens, from a panel of 9 typical candidate genes, the expression of ACTB, HMBS, HPRT1, RPL13, RPL32, 18SrRNA, TBP, TFRC, and YWHAZ was determined in the lung and heart (right ventricle) of both healthy and cold-induced pulmonary hypertensive chickens at 42 d of age. The BestKeeper, geNorm, and NormFinder software programs were used to analyze this set of genes. Also, the ratio of right ventricle to the total ventricle was used as an index of induced pulmonary hypertension, which increased in the cold-treated chickens compared to the control at 42 d of age. Candidate reference genes ranking in the lung of pulmonary hypertensive chickens vs. healthy individuals included RPL13, YWHAZ, HMBS, ACTB, HPRT1, TFRC, RPL32, 18SrRNA, and TBP; those in the heart were YWHAZ, RPL13, HMBS, ACTB, HPRT1, TBP, 18SrRNA, TFRC, and RPL32; and those in the heart-lung combination included RPL13, YWHAZ, HMBS, HBRT1, TFRC, ACTB, 18SrRNA, RPL32, and TBP. The overall results showed that the most stable genes are YWHAZ, RPL13, HMBS, ACTB, HBRT1, TFRC, TBP, RPL32, and 18SrRNA, respectively. In addition, the combination of YWHAZ, RPL13, and HMBS is recommended as the reference gene panel for more accurate quantitative data normalization of heart or lung in the chicken pulmonary hypertension.