Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1-and M2-polarized macrophage
Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1-and M2-polarized macrophage
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在小鼠 M1 和 M2 极化巨噬细胞中通过 RT-qPCR 进行 mRNA 表达分析的参考基因选择和验证
DOI：
10.1007/s11033-020-05372-z
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发表时间：
2020-03-19
影响因子：
2.8
通讯作者：
Zeng, Lingyu
中科院分区：
文献类型：
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作者：
Ju, Wen;Sun, Tiantian;Zeng, Lingyu
小鼠骨髓源性巨噬细胞（M0）和M1-和m2极化巨噬细胞被广泛用作极化巨噬细胞相关分子机制分析的实验室模型。基于内参基因归一化的RT-qPCR基因表达分析是探索其分子机制的有力途径。但对这些细胞模型中的内参基因知之甚少。因此，本研究的目的是鉴定这些巨噬细胞类型中的内参基因。使用Limma线性模型方法从微阵列数据中选择小鼠骨髓源性和极化巨噬细胞中的候选内参基因，并使用BestKeeper、NormFinder、GeNorm、Delta CT方法和RefFinder五种算法确定稳定性值进行评估。最后，通过检测细胞系中三个重要的免疫和炎症基因（NLRP1， IL-1 β和tnf - α）来验证所选择的稳定内参基因。我们的研究清楚地表明，Ubc后接Eef1a1和B2m分别被认为是小鼠骨髓源性和极化巨噬细胞中基因表达分析的三个理想内参基因。当使用三个稳定性较强的内参基因进行验证时，获得了IL-1 β、tnf - α和NLRP1基因表达水平的较大变化，这清楚地证明了RT-qPCR分析需要仔细选择内参基因。为了保证qPCR分析的准确性，需要在巨噬细胞和极化巨噬细胞中使用Ubc而不是Actb或Gusb使mRNA表达水平正常化。
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-1 beta, and TNF-alpha) 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-1 beta, TNF-alpha 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.