College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; College of Animal Science and Technology, Hebei North University, Zhangjiakou, Hebei 075000, China.
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Gene. 2022 Jul 1;830:146502. doi: 10.1016/j.gene.2022.146502. Epub 2022 Apr 25.
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.
内参基因的稳定性对于实时荧光定量 PCR（qRT-PCR）中基因表达结果的可靠性至关重要。不合适的内参基因可能导致不准确的结果，甚至错误的结论。本研究旨在鉴定用于分析体外牛原代前体脂肪细胞（BPP）中增殖相关和诱导分化基因表达的稳定内参基因。在这项研究中，通过三种不同的算法（geNorm、NormFinder 和 BestKeeper）研究了 16 个候选内参基因（GAPDH、ACTB、PPIA、LRP10、HPRT1、YWHAZ、B2M、TBP、EIF3K、RPS9、UXT、18S rRNA、RPLP0、MARVELD、EMD 和 RPS15A）在 BPP 增殖和分化阶段进行 qRT-PCR 的稳定性。使用两个标记基因 PCNA 和 LPL 的表达来分别确定候选内参基因（RGs）在增殖和分化阶段的有效性。结果表明，GAPDH 和 RPS15A 是牛原代前体脂肪细胞增殖过程中最稳定的 RG，而 PPIA 是最不稳定的内参基因。RPLP0 和 EIF3K 是牛原代前体脂肪细胞分化诱导过程中最稳定的 RG，而 GAPDH 是最不稳定的内参基因。本研究为随后使用 qRT-PCR 研究 BPP 体外增殖和分化的机制奠定了基础。
