BACKGROUND For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data. RESULTS The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt +/- SD, 23.66 +/- 0.86) and RPL32 (18.65 +/- 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (p = 0.004); no change was observed using GAPDH/ACTB (p > 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (p < 0.03). CONCLUSION PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.

中文翻译：

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PSMB2 和 RPL32 是用于标准化支气管肺泡细胞中基因表达谱的合适分母。

背景 为了定量逆转录酶-聚合酶链反应 (qRT-PCR) 的准确性，需要使用合适的参考基因进行标准化。迄今为止，尚未验证任何参考基因可用于支气管肺泡 (BAL) 细胞的表达研究。本研究的目的是鉴定在 BAL 细胞中具有稳定 mRNA 表达的基因，而不论性别、吸烟、BAL 细胞组成、肺病理、治疗；并评估参考基因对目标基因表达数据的影响。结果 通过 qRT-PCR 在来自 71 名不同肺部疾病受试者的 BAL 细胞中研究了 10 种管家基因（ACTB、ARF1、CANX、G6PD、GAPDH、GPS1、GNB2L1、PSMB2、PSMD2、RPL32）的 mRNA 表达。这些分析在来自 63 名结节病患者和 17 名对照受试者的独立 BAL 队列中得到验证。使用二阶导数法计算表达值（CTt）；在等效性测试中，应用小程序 BestKeeper、geNorm 和 NormFinder 来研究基因表达的稳定性。在研究的基因中，PSMB2 (CTt +/- SD, 23.66 +/- 0.86) 和 RPL32 (18.65 +/- 0.92) 是最稳定的；无论评估变量如何，两者都在来自平行研究队列的 BAL 样本中不断表达。最后，为了证明传统 (ACTB/GAPDH) 和新型 (PSMB2/RPL32) 参考基因作为分母的效果，在结节 BAL 细胞中研究了两种已知与结节病相关的细胞因子的表达。虽然用 PSMB2/RPL32 标准化导致 IFNG mRNA 表达升高（p = 0.004）；使用 GAPDH/ACTB 没有观察到变化（p > 0.05）。仅当 PSMB2/RPL32 用作分母时才观察到 CCL2 mRNA 上调（p < 0.03）。因此，结论 PSMB2 和 RPL32 是使结节病和其他间质性肺病 BAL 细胞中的 qRT-PCR 正常化的合适参考基因。