Kriegova Eva, Arakelyan Arsen, Fillerova Regina, Zatloukal Jaromir, Mrazek Frantisek, Navratilova Zdenka, Kolek Vitezslav, du Bois Roland M, Petrek Martin
Department of Immunology, Palacky University, The Czech Republic.
BMC Mol Biol. 2008 Jul 31;9:69. doi: 10.1186/1471-2199-9-69.
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.
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).
PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.
为确保定量逆转录聚合酶链反应（qRT-PCR）的准确性，需要使用合适的内参基因进行标准化。迄今为止，尚未有内参基因在支气管肺泡（BAL）细胞表达研究中得到验证。本研究的目的是确定在BAL细胞中mRNA表达稳定的基因，且不受性别、吸烟、BAL细胞组成、肺部病理、治疗的影响；并评估内参基因对靶基因表达数据的影响。
通过qRT-PCR研究了来自71例患有各种肺部疾病受试者的BAL细胞中10个管家基因（ACTB、ARF1、CANX、G6PD、GAPDH、GPS1、GNB2L1、PSMB2、PSMD2、RPL32）的mRNA表达。分析在来自63例结节病患者和17例对照受试者的独立BAL队列中得到验证。使用二阶导数法计算表达值（CTt）；应用等效性检验、BestKeeper小程序、geNorm和NormFinder来研究基因表达稳定性。在所研究的基因中，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，二者在平行研究队列的BAL样本中均持续表达。最后，为证明传统（ACTB/GAPDH）和新型（PSMB2/RPL32）内参基因作为分母的影响，在结节病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。用PSMB2/RPL32进行标准化导致IFNG mRNA表达升高（p = 0.004）；使用GAPDH/ACTB则未观察到变化（p>0.05）。仅当使用PSMB2/RPL32作为分母时观察到CCL2 mRNA上调（p < 0.03）。
因此，PSMB2和RPL32是结节病及其他间质性肺疾病中用于标准化BAL细胞qRT-PCR的合适内参基因。
