Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str, 25, 07743 Jena, Germany.
BMC Mol Biol. 2010 Dec 1;11:90. doi: 10.1186/1471-2199-11-90.
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.
对于使用定量实时 RT-PCR（RT-qPCR）分析基因表达的任何功能研究，可靠的参考基因是至关重要的前提。然而，很少有研究将参考基因的选择和评估纳入其中。迄今为止，还没有针对 THP-1 单核细胞分化的参考基因得到验证。在这里，我们报告了在佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）诱导 THP-1 单核细胞分化为巨噬细胞过程中，选择经过验证的参考基因的情况。
在 PMA 诱导的 THP-1 单核细胞向巨噬细胞分化的六天过程中的多个时间点，通过 RT-qPCR 测量了 21 个预选潜在参考基因的 mRNA 表达。根据表达稳定性进行了排名计算。计算使用基于 Microsoft Excel 的小程序 GeNorm、NormFinder 和 BestKeeper 进行。我们的结果表明 ACTB（β-肌动蛋白）（Cq ± SD，14.1 ± 0.3）和 RPL37A（核糖体蛋白 L37a）（14.5 ± 0.3）是最稳定的基因。而其他常用的参考基因，如 GAPDH（甘油醛-3-磷酸脱氢酶）（20.8 ± 0.8）或 G6PD（葡萄糖-6-磷酸脱氢酶）（16.1 ± 1.0）则被认为不够可靠，因此不适合用作参考基因。这些发现通过研究已知在单核细胞向巨噬细胞分化过程中受调控的巨噬细胞清道夫受体 CD36 的 mRNA 表达得到了验证。使用 ACTB 和 RPL37A 作为参考基因，可以显著地证明 CD36 的深度调节，而使用 G6PD 则导致 CD36 的调节作用明显减弱。
因此，建议在 THP-1 单核细胞分化过程中，使用 ACTB 和 RPL37A 对任何基于实时 PCR 的表达数据进行归一化。
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