Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China.
Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China.
Mol Biol Rep. 2020 Apr;47(4):2735-2748. doi: 10.1007/s11033-020-05372-z. Epub 2020 Mar 19.
Murine bone marrow-derived macrophages (M0) and M1- and M2-polarized macrophages are being widely used as a laboratory model for polarized macrophages related molecular mechanism analysis. Gene expression analysis based on reference gene normalization using RT-qPCR was a powerful way to explore the molecular mechanism. But little is known about reference genes in these cell models. So, the goal of this study was to identify reference genes in these types of macrophages. Candidate reference genes in murine bone marrow-derived and polarized macrophages were selected from microarray data using Limma linear model method and evaluated by determining the stability value using five algorithms: BestKeeper, NormFinder, GeNorm, Delta CT method, and RefFinder. Finally, the selected stable reference genes were validated by testing three important immune and inflammatory genes (NLRP1, IL-1β, and TNF-α) in the cell lines. Our study has clearly shown that Ubc followed by Eef1a1 and B2m respectively were recognized as the three ideal reference genes for gene expression analysis in murine bone marrow-derived and polarized macrophages. When three reference genes with strong different stability were used for validation, a large variation of a gene expression level of IL-1β, TNF-α and NLRP1 were obtained which provides clear evidence of the need for careful selection of reference genes for RT-qPCR analysis. Normalization of mRNA expression level with Ubc rather than Actb or Gusb by qPCR in macrophages and polarized macrophages is required to ensure the accuracy of the qPCR analysis.
小鼠骨髓来源的巨噬细胞（M0）以及 M1 和 M2 极化的巨噬细胞被广泛用作与极化巨噬细胞相关的分子机制分析的实验室模型。基于 RT-qPCR 的参考基因标准化的基因表达分析是探索分子机制的有力方法。但是，对于这些细胞模型中的参考基因知之甚少。因此，本研究的目的是鉴定这些类型的巨噬细胞中的参考基因。使用 Limma 线性模型方法从微阵列数据中选择了小鼠骨髓来源和极化巨噬细胞中的候选参考基因，并通过使用五种算法（BestKeeper、NormFinder、GeNorm、Delta CT 方法和 RefFinder）确定稳定性值来进行评估。最后，通过测试细胞系中的三个重要免疫和炎症基因（NLRP1、IL-1β和 TNF-α）来验证所选的稳定参考基因。我们的研究清楚地表明，Ubc 随后分别由 Eef1a1 和 B2m 确定为小鼠骨髓来源和极化巨噬细胞中基因表达分析的三个理想参考基因。当使用三个稳定性差异较大的参考基因进行验证时，IL-1β、TNF-α 和 NLRP1 的基因表达水平会发生很大变化，这为 RT-qPCR 分析中参考基因的仔细选择提供了明确的证据。在巨噬细胞和极化巨噬细胞中，通过 qPCR 使用 Ubc 而不是 Actb 或 Gusb 对 mRNA 表达水平进行归一化是确保 qPCR 分析准确性所必需的。
