Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Roshini Kalagara; Weimin Gao; Honor L Glenn; Colleen Ziegler; Laura Belmont; Deirdre R Meldrum

doi:10.1093/biomethods/bpw005

Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Biol Methods Protoc. 2016 Dec 27;1(1):bpw005. doi: 10.1093/biomethods/bpw005. eCollection 2016 Mar.

Authors

Roshini Kalagara¹, Weimin Gao¹, Honor L Glenn¹, Colleen Ziegler¹, Laura Belmont¹, Deirdre R Meldrum¹

Affiliation

¹ Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.

Abstract

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using C_t value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

Keywords: J774A.1; PCR; housekeeping genes; inflammation; murine; standardization.