Spinsanti Giacomo, Panti Cristina, Bucalossi Daniela, Marsili Letizia, Casini Silvia, Frati Francesco, Fossi Maria Cristina
Department of Evolutionary Biology, University of Siena, Via A. Moro 2, 53100 Siena (SI), Italy.
Aquat Toxicol. 2008 May 1;87(3):178-86. doi: 10.1016/j.aquatox.2008.01.018. Epub 2008 Feb 8.
Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies.
定量实时聚合酶链反应（qRT-PCR）是一种用于检测生物样品中mRNA表达的有效分子技术。其灵敏度能够对基因转录调控中的微小变化进行定量，但严格依赖于标准化程序中所采用的方法。相对定量可确定多个样品中基因稳态mRNA水平的变化，并通过与一种或多种内参RNA的水平进行比较来评估。在这种情况下，选择组成型表达的对照基因（其转录不受污染物影响）对于该技术的可靠性似乎至关重要。在本研究中，源自小斑原海豚（Stenella coeruleoalba）皮肤活检的成纤维细胞培养物，暴露于具有内分泌干扰能力（EDCs）的不同化合物混合物（有机氯（OCs）、多溴二苯醚（PBDEs）和17β-雌二醇）的浓度递增环境中6小时。使用qRT-PCR分析检测了10个常见管家基因在暴露的细胞培养物中转录本的表达情况，并使用两个Excel小程序geNorm和NormFinder对原始数据进行了分析。编码SDHA、GAPDH和YWHAZ的基因分别似乎是OC、PBDE和17β-雌二醇处理最可靠的对照基因。这些结果清楚地表明，即使是广泛分布的对照基因的转录也可受到不同处理的调控，并突显了在毒理学研究中仔细选择最佳管家基因的重要性。
