Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells

Guoqiang Ren, Morten Juhl, Qiuyue Peng, Trine Fink, Simone Riis Porsborg*

*Corresponding author for this work

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For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.

Original languageEnglish
JournalImmunology & Cell Biology
Issue number10
Pages (from-to)822-829
Number of pages8
Publication statusPublished - Nov 2022

Bibliographical note

© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.


  • Interferon-γ
  • Lipopolysaccharide
  • M1macrophage-like cells
  • RT-qPCR
  • Reference gene
  • THP-1 Cells


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