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BMC Immunology

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Open Access 01-12-2010 | Research article

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

Authors: Armin P Piehler, Runa M Grimholt, Reidun Øvstebø, Jens P Berg

Published in: BMC Immunology | Issue 1/2010

Abstract

Background

Gene expression in lipopolysaccharide (LPS)-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR) using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta-actin) as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system.

Results

Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B), B2M (beta-2-microglobulin) and PPIA (cyclophilin A) as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein) and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha) and IL10 (interleukin 10) expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH.

Conclusions

Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

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Title: Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes
Authors: Armin P Piehler
Runa M Grimholt
Reidun Øvstebø
Jens P Berg
Publication date: 01-12-2010
Publisher: BioMed Central
Published in: BMC Immunology / Issue 1/2010
Electronic ISSN: 1471-2172
DOI: https://doi.org/10.1186/1471-2172-11-21

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