BACKGROUND Quantitative PCR (qPCR) is a powerful tool that is particularly well-suited to measure mRNA levels in clinical samples, especially those with relatively low cell counts. However, a caveat of this approach is that reliable, stably expressed reference (housekeeping) genes are vital in order to ensure reproducibility and appropriate biological inference. In this study, we evaluated the expression stability of six reference genes in peripheral blood mononuclear cells (PBMCs) and isolated CD3+ T-cells from young and old adults (n = 10), following ex vivo stimulation with mock (unstimulated) or live influenza virus. Our genes included: β-actin (ACTB), glyercaldehyde-3-phostphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13a), ribosomal protein S18 (RPS18), succinate dehydrogenase complex flavoprotein subunit A (SDHA), and ubiquitin-conjugating enzyme E2D2 (UBE2D2). RESULTS Reference gene expression varied significantly depending on cell type and stimulation conditions, but not age. Using the comparative ΔCt method, and the previously published software BestKeeper, NormFinder, and geNorm, we show that in PBMCs and T-cells, UBE2D2 and RPS18 were the most stable reference genes, followed by ACTB; however, the expression of UBE2D2 and RPS18 was found to increase with viral stimulation in isolated T-cells, while ACTB expression did not change significantly. No age-related differences in stability were observed for any gene CONCLUSIONS: This study suggests the use of a combination of UBE2D2, RPS18, and ACTB for the study of influenza responses in PBMCs and T-cells, although ACTB alone may be the most optimal choice if choosing to compare target gene expression before and after viral stimulation. Both GAPDH and RPL13a were found to be poor reference genes and should be avoided for studies of this nature.

中文翻译：

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用于定量活流感病毒刺激的人T细胞和PBMC中mRNA的可靠参考基因。

背景技术定量PCR（qPCR）是一种功能强大的工具，特别适合于测量临床样品中的mRNA水平，尤其是那些细胞计数相对较低的样品。但是，此方法的一个警告是，可靠，稳定表达的参考（管家）基因对于确保可重复性和适当的生物学推断至关重要。在这项研究中，我们通过模拟（未刺激）或活流感离体刺激后，评估了六个参考基因在外周血单个核细胞（PBMC）和来自年轻人和老年人（n = 10）的分离的CD3 + T细胞中的表达稳定性病毒。我们的基因包括：β-肌动蛋白（ACTB），甘油三醛-磷酸脱氢酶（GAPDH），核糖体蛋白L13a（RPL13a），核糖体蛋白S18（RPS18），琥珀酸脱氢酶复合物黄素蛋白亚基（SDHA），和泛素结合酶E2D2（UBE2D2）。结果参考基因的表达随细胞类型和刺激条件而变化，但与年龄无关。使用比较性ΔCt方法以及先前发布的软件BestKeeper，NormFinder和geNorm，我们显示在PBMC和T细胞中，UBE2D2和RPS18是最稳定的参考基因，其次是ACTB。然而，在分离的T细胞中，UBE2D2和RPS18的表达随病毒刺激而增加，而ACTB的表达没有明显改变。结论：本研究建议使用UBE2D2，RPS18和ACTB的组合研究PBMC和T细胞中的流感反应，尽管如果选择比较病毒刺激前后的靶基因表达，单独使用ACTB可能是最佳选择。发现GAPDH和RPL13a均为不良参考基因，应避免用于此类研究。