Analysis of stability of reference genes for qPCR in bovine preadipocytes during proliferation and differentiation in vitro.

ABSTRACT

The stability of internal reference genes is crucial to the reliability of gene expression results using real-time fluorescence quantitative PCR (qRT-PCR). Inappropriate reference genes may lead to inaccurate results or even wrong conclusions. This study aims to identify stable reference genes for analyzing the expression of proliferation-related and differentiation-inducing genes in bovine primary preadipocytes (BPPs) in vitro. In this study, the stability of 16 candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K, RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) for qRT-PCR at proliferation and differentiation stages of BPPs was investigated by three different algorithms (geNorm, NormFinder and BestKeeper). The expression of two marker genes, PCNA and LPL, was used to determine the validity of the candidate reference genes (RGs) at the proliferation and differentiation stages, respectively. The results showed that GAPDH and RPS15A were the most stable RGs in the proliferation of bovine primary preadipocyte, while PPIA was the least stable internal reference gene. RPLP0 and EIF3K were the most stable RGs in the differentiation induction of bovine primary preadipocyte, while GAPDH was the least stable internal reference gene. This study of RGs laid the foundation for subsequent research into the mechanism of proliferation and differentiation of BPPs in vitro using qRT-PCR.