Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages
Open Access
- 1 December 2010
- journal article
- Published by Springer Science and Business Media LLC in BMC Molecular Biology
- Vol. 11 (1), 90
- https://doi.org/10.1186/1471-2199-11-90
Abstract
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). 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 (β-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. Consequently, it is recommended to normalize any real-time PCR-based expression data obtained during THP-1 monocyte differentiation using ACTB and RPL37A.Keywords
This publication has 36 references indexed in Scilit:
- Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes.BMC Immunology, 2010
- Validation of suitable internal control genes for expression studies in agingMechanisms of Ageing and Development, 2009
- Uptake of Oxidized Low Density Lipoprotein by CD36 Occurs by an Actin-dependent Pathway Distinct from MacropinocytosisPublished by Elsevier BV ,2009
- Selection of reliable reference genes for gene expression studies in peach using real-time PCRBMC Molecular Biology, 2009
- Identification of valid reference genes during the differentiation of human myoblastsBMC Molecular Biology, 2009
- Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognosesBMC Cancer, 2009
- Genomic selection of reference genes for real-time PCR in human myocardiumBMC Medical Genomics, 2008
- Macrophage diversity in renal injury and repairJCI Insight, 2008
- Performance comparison of two microarray platforms to assess differential gene expression in human monocyte and macrophage cellsBMC Genomics, 2008
- Transcriptional diversity during monocyte to macrophage differentiationImmunology Letters, 2008