A comparative assessment of reference genes in mouse brown adipocyte differentiation and thermogenesis in vitro

ABSTRACT

Adipogenic differentiation and thermogenesis in brown adipose tissue (BAT) undergo dynamic processes, altering phenotypes and gene expressions. Proper reference genes in gene expression analysis are crucial to mitigate experimental variances and ensure PCR efficacy. Unreliable reference genes can lead to erroneous gene expression quantification, resulting in data misinterpretation. This study focused on identifying suitable reference genes for mouse brown adipocyte research, utilizing brown adipocytes from the Ucp1-luciferase ThermoMouse model. Comparative analysis of gene expression data under adipogenesis and thermogenesis conditions was conducted, validating 13 housekeeping genes through various algorithms, including DeltaCq, BestKeeper, geNorm, Normfinder, and RefFinder. Tbp and Rer1 emerged as optimal references for Ucp1 and Pparg expression in brown adipogenesis, while Tbp and Ubc were ideal for the expression analysis of these target genes in thermogenesis. Conversely, certain conventional references, including Actb, Tubb5, and Gapdh, proved unstable as reference genes under both conditions. These findings stress the critical consideration of reference gene selection in gene expression analysis within specific biological systems to ensure accurate conclusions.

KEYWORDS:

• Reference gene
• brown adipocytes
• Ucp1
• thermogenesis
• adipocyte differentiation

Acknowledgments

We thank all the lab members for their thoughtful feedback on this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The reference sequences used for designing primers in this study can be found in the NCBI Reference Sequences (NM007393.5; NM009735.3; NM001289726.2; NM010368.2; NM013556.2; NM008828.3; NM026395.2; NM009438.5; NM011296.3; NM013684.3; NM011655.5; NM019639.4; NM023281.1; NM009463.3; NM001127330.3).

Accession codes

The code used to generate the figures and tables and run the analysis in this study is open-source (GPL-3) and freely available at https://github.com/BCMSLab.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21623945.2024.2330355

Additional information

Funding

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea [RS-2023-00219399 and RS- 2023-00274727] and by a Commercialization Promotion Agency for R&D Outcomes [COMPA] grant funded by the Korean government [MSIT] [1711173796].