Using RNA-Seq to Identify Reference Genes of the Transition from Brown to White Adipose Tissue in Goats

SIMPLE SUMMARY: Brown adipose tissue (BAT) plays important roles in unique non-shivering thermogenesis. It is necessary to select reference genes during the transition process from brown (BAT) to white adipose tissue (WAT) for Quantitative PCR (qPCR) analysis. In this study, CTNNB, PFDN5 and EIF3M, selected from RNA sequencing data, were the most suitable reference genes. The present study provides a detailed analysis of the expression stability of reference genes for the study of gene expression profiling during the transition process from BAT to WAT. ABSTRACT: Brown adipose tissues have unique non-shivering thermogenesis functions, can be found in newborn ruminate animals, and then are gradually replaced by white adipose tissues in adulthood. For the purpose of exploring the intrinsic mechanism underlying the conversion process from brown (BAT) to white adipose tissue (WAT), it is necessary to utilize Quantitative PCR (qPCR) to study gene expression profiling. In this study, we identified reference genes that were consistently expressed during the transformation from goat BAT to WAT using RNA-seq data. Then, twelve genes were evaluated as candidate reference genes for qPCR in goat perirenal adipose tissue using three tools (geNorm, Normfinder, and BestKeeper). In addition, the selected reference genes were used to normalize the gene expression of PGC-1α and GPAT4. It was found that traditional reference genes, such as GAPDH, RPLP0, HPRT1, and PPIA were not suitable for target gene normalization. In contrast, CTNNB, PFDN5, and EIF3M, selected from RNA sequencing data, showed the least variation and were recommended as the best reference genes during the transformation from BAT to WAT.