Housekeeping gene expression variability in differentiating and non-differentiating 3T3-L1 cells

Danang Dwi Cahyadi; Tomoko Warita; Nanami Irie; Kana Mizoguchi; Jiro Tashiro; Yoshinao Z Hosaka; Katsuhiko Warita

doi:10.1080/21623945.2023.2235081

Housekeeping gene expression variability in differentiating and non-differentiating 3T3-L1 cells

Adipocyte. 2023 Dec;12(1):2235081. doi: 10.1080/21623945.2023.2235081.

Authors

Danang Dwi Cahyadi^{1

2}, Tomoko Warita³, Nanami Irie⁴, Kana Mizoguchi⁴, Jiro Tashiro¹, Yoshinao Z Hosaka⁵, Katsuhiko Warita^{1

6}

Affiliations

¹ Joint Graduate School of Veterinary Sciences, Tottori University, Tottori, Tottori, Japan.
² Division of Anatomy Histology and Embryology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, West Java, Indonesia.
³ Department of Biomedical Sciences, School of Biological and Environmental Sciences, Kwansei Gakuin University, Sanda, Hyogo, Japan.
⁴ Graduate School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo, Japan.
⁵ Department of Bioresource Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Fukuoka, Japan.
⁶ Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Tottori, Japan.

Abstract

Normalization is a crucial step in gene expression analysis to avoid misinterpretation. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of 10 candidate housekeeping genes in non-differentiated (ND) and differentiated (DI) 3T3-L1 cells on days 5 and 10. We used geNorm, NormFinder, BestKeeper, RefFinder, and the ∆Ct method to evaluate expression stability. The findings revealed that (1) the expression levels of the reference genes changed over time, even in non-differentiating cells, and (2) peptidylprolyl isomerase A (Ppia) and TATA box-binding protein (Tbp) were stable reference genes for 10 days in both undifferentiated and differentiated 3T3-L1 cells. Notably, the expression of known reference genes in non-differentiating cells was altered throughout the experiment.

Keywords: adipocytes; differentiation; gene expression; normalization; reference gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

3T3-L1 Cells
Animals
Cell Differentiation / genetics
Gene Expression Profiling*
Genes, Essential*
Mice

Grants and funding

The work was supported by the Japan Society for the Promotion of Science [JSPS KAKENHI JP19H03514] to K.W. in a Grant-in-Aid for Scientific Research (B); Japan Society for the Promotion of Science [JSPS KAKENHI JP20K06411] to Y.Z.H. in a Grant-in-Aid for Scientific Research (C).