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Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1- and M2-polarized macrophage

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Abstract

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.

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The data and materials used to support the findings in this study are included in the article.

Abbreviations

BMDM or M0:

Bone marrow-derived macrophage

RT-qPCR:

Real-time quantitative PCR

M1:

Classically activated macrophages

M2:

Alternatively activated macrophages

RG:

Reference gene

STDEV:

Standard deviation

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Funding

This study was supported in part by National Natural Science Foundation of China under Grant [Nos. 81700178, 31872795 and 81570096]; Natural Science Foundation of Jiangsu Province under Grant [No. BK20170259]; Jiangsu Provincial Key Research and Development Program under Grant [No. BE2018637]; Jiangsu Province’s Key Provincial Talents Program under Grant [No. ZDRCA2016054]; China Postdoctoral Science Foundation Grant [No. 2018M632380]; and Jiangsu Postdoctoral Science Foundation under Grant [No. 1701064B] for purchasing reagents, testing fee, and publication charge.

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Author notes

  1. Wen Ju, Wenyi Lu and Tiantian Sun contributed equally to this work.

Authors and Affiliations

  1. Blood Diseases Institute, Xuzhou Medical University, Xuzhou, 221002, China

    Wen Ju, Kunming Qi, Kailin Xu, Jianlin Qiao & Lingyu Zeng

  2. Key Laboratory of Bone Marrow Stem Cell, Xuzhou, 221002, Jiangsu Province, China

    Wen Ju, Tiantian Sun, Wenyi Lu, Alhaji Osman Smith, Yurong Bao, Seyram Yao Adzraku, Kunming Qi, Kailin Xu, Jianlin Qiao & Lingyu Zeng

  3. Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China

    Wen Ju, Tiantian Sun, Wenyi Lu, Alhaji Osman Smith, Yurong Bao, Seyram Yao Adzraku, Kunming Qi, Kailin Xu, Jianlin Qiao & Lingyu Zeng

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  1. Wen Ju
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Contributions

Conceptualization, AOS; Data curation, WJ and AOS; Formal analysis, KQ; Funding acquisition, WJ; Investigation, WJ, TS, WL, KQ and LZ; Methodology, TS, WL, YB, SYA and KQ; Project administration, WL; Resources, JQ and LZ; Software, TS, WL, YB and SYA; Supervision, LZ; Validation, WJ, KX, JQ and LZ; Visualization, JQ and LZ; Writing—original draft, WJ; Writing—review & editing, AOS, SYA, KX, JQ and LZ.

Corresponding authors

Correspondence to Jianlin Qiao or Lingyu Zeng.

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All authors approved the submission of the manuscript.

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All animal care and procedures were per the ethical standards, approved by Jiangsu Society for Animal Welfare, China (Acceptance number: XZMC20130226) and Science and Technology Department of Jiangsu Province, China (Acceptance number: SYXK(SU)-2015-0030 and SCXK(SU)-2015-0009).

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Ju, W., Sun, T., Lu, W. et al. Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1- and M2-polarized macrophage. Mol Biol Rep 47, 2735–2748 (2020). https://doi.org/10.1007/s11033-020-05372-z

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