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Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages.

ABSTRACT: BACKGROUND: Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA). RESULTS: The mRNA expression of 21 preselected potential reference genes was measured by RT-qPCR at several time-points over six days of PMA-induced THP-1 monocyte-to-macrophage differentiation. A ranking according to expression stability was calculated. Calculations were performed using Microsoft Excel-based applets GeNorm, NormFinder and BestKeeper. Our results indicated ACTB (?-actin) (Cq ± SD, 14.1 ± 0.3) and RPL37A (ribosomal protein L37a) (14.5 ± 0.3) as the most stable genes. While other frequently used reference genes such as GAPDH (glycereraldehyde-3-phosphate dehydrogenase) (20.8 ± 0.8) or G6PD (glucose-6-phophate dehydrogenase) (16.1 ± 1.0) were found to be not as reliable and were therefore unsuited for use as reference genes. These findings were validated by investigating mRNA expression of macrophage scavenger receptor CD36, known to be regulated during monocyte-to-macrophage differentiation. Using ACTB and RPL37A as reference genes a profound and significant regulation of CD36 could be demonstrated, while use of G6PD resulted in a much less pronounced apparent regulation of CD36. CONCLUSION: Consequently, it is recommended to normalize any real-time PCR-based expression data obtained during THP-1 monocyte differentiation using ACTB and RPL37A.

SUBMITTER: Maess MB

PROVIDER: S-EPMC3002353 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages.

Maess Marten B MB Sendelbach Stefanie S Lorkowski Stefan S

BMC molecular biology 20101201

<h4>Background</h4>Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by ...[more]

PMID: 21122122

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Project description:BackgroundMacrophages are key players in the initiation, perpetuation and regulation of both innate and adaptive immune responses. They largely perform these roles through modulation of the expression of genes, especially those encoding cytokines. Murine bone marrow derived macrophages (BMDMs) are commonly used as a model macrophage population for the study of immune responses to pro-inflammatory stimuli, notably lipopolysaccharide (LPS), which may be pertinent to the human situation. Evaluation of the temporal responses of LPS stimulated macrophages is widely conducted via the measurement of gene expression levels by RT-qPCR. While providing a robust and sensitive measure of gene expression levels, RT-qPCR relies on the normalisation of gene expression data to a stably expressed reference gene. Generally, a normalisation gene(s) is selected from a list of "traditional" reference genes without validation of expression stability under the specific experimental conditions of the study. In the absence of such validation, and given that many studies use only a single reference gene, the reliability of data is questionable.ResultsThe stability of expression levels of eight commonly used reference genes was assessed during the peak (6 h) and resolution (24 h) phases of the BMDM response to LPS. Further, this study identified two additional genes, which have not previously been described as reference genes, and the stability of their expression levels during the same phases of the inflammatory response were validated. Importantly, this study demonstrates that certain "traditional" reference genes are in fact regulated by LPS exposure, and, therefore, are not reliable candidates as their inclusion may compromise the accuracy of data interpretation. Testament to this, this study shows that the normalisation of gene expression data using an unstable reference gene greatly affects the experimental data obtained, and, therefore, the ultimate biological conclusions drawn.ConclusionThis study reaffirms the importance of validating reference gene stability for individual experimental conditions. Given that gene expression levels in LPS stimulated macrophages is routinely used to infer biological phenomena that are of relevance to human conditions, verification of reference gene expression stability is crucial.

Dataset Information

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages.

Publications

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages.

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