Identification of Reference Genes for Quantitative Gene Expression Studies in Three Tissues of Japanese Quail

Abstract

RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species.