Sanabria Natasha M, Gulumian Mary
National Institute for Occupational Health, Johannesburg, South Africa.
Haematology and Molecular Medicine Department, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.
J Nanobiotechnology. 2017 Oct 10;15(1):72. doi: 10.1186/s12951-017-0299-9.
RT-qPCR is routinely used in expression profiling of toxicity pathway genes. However, genetic and molecular level studies used to determine, understand and clarify potential risks of engineered nanomaterials (ENMs) are still incomplete. Concerns regarding possible interference caused by intracellular ENMs during analyses have been raised. The aim of this study was to verify a qPCR procedure for gene expression assays, which can be used in toxicity and exposure assessments.
Amplification of ten reference genes was performed to test the expression stability. A preliminary study was performed on RNA from BEAS-2B cells that had been treated with AuNPs. Also, a reference total RNA standard from ten cell lines was spiked with various amounts of the same AuNP. This treatment mimics exposure assessment studies, where assay-interference may be caused by intracellular residual ENMs still being present in the biological samples (during and after isolation/purification procedures). Both types of RNA samples were reverse transcribed and then amplified by qPCR. The qPCR-related software and statistical programs used included BestKeeper, NormFinder, REST and qBase+. These results proved that using standard qPCR analysis and statistical programs should not be the only procedure applied to verify the assay for gene expression assessment related to ENMs. A comparison of SYBR Green to EVA Green was discussed, in addition to a comparison to the latest reports regarding the influence of ENM thermal conductivity, surface interactions with ENMs, effects of ENM size and charge, as well as, the limit of detection in a qPCR assay.
AuNPs have the potential to interfere with the assay mechanism of RT-qPCR, thus, assay verification is required for AuNP-related gene expression studies used to evaluate toxicity. It is recommended to use HSP90 and YWHAZ as reference genes, i.e. these were the most stable in our study, irrespective of the source of the RNA, or, the point at which the AuNPs interacted with the assay. This report describes steps that can be utilised to generate a suitable method for gene expression studies associated with toxicity testing of various ENMs. For example, RNA standards that have been spiked with known amounts of ENMs should be run in conjunction with the unknown samples, in order to verify any RT-qPCR assay and determine the degree of error.
逆转录定量聚合酶链反应（RT-qPCR）常用于毒性通路基因的表达谱分析。然而，用于确定、理解和阐明工程纳米材料（ENM）潜在风险的遗传和分子水平研究仍不完整。人们对分析过程中细胞内ENM可能造成的干扰表示担忧。本研究的目的是验证一种用于基因表达检测的qPCR方法，该方法可用于毒性和暴露评估。
对十个参考基因进行扩增以测试表达稳定性。对用金纳米颗粒（AuNP）处理过的BEAS-2B细胞的RNA进行了初步研究。此外，向来自十个细胞系的参考总RNA标准品中加入不同量的相同AuNP。这种处理模拟了暴露评估研究，在该研究中，生物样品中（在分离/纯化过程期间和之后）仍存在的细胞内残留ENM可能会导致检测干扰。两类RNA样品均进行逆转录，然后通过qPCR扩增。使用的qPCR相关软件和统计程序包括BestKeeper、NormFinder、REST和qBase+。这些结果证明，使用标准qPCR分析和统计程序不应是验证与ENM相关的基因表达评估检测的唯一方法。除了与关于ENM热导率、与ENM的表面相互作用、ENM大小和电荷的影响以及qPCR检测中的检测限的最新报告进行比较外，还讨论了SYBR Green与EVA Green的比较。
AuNP有可能干扰RT-qPCR的检测机制，因此，用于评估毒性的与AuNP相关的基因表达研究需要进行检测验证。建议使用热休克蛋白90（HSP90）和14-3-3蛋白ζ（YWHAZ）作为参考基因，即，在我们的研究中，无论RNA来源如何，或者AuNP与检测相互作用的时间点如何，这两个基因都是最稳定的。本报告描述了可用于生成与各种ENM毒性测试相关的基因表达研究的合适方法的步骤。例如，应将加入已知量ENM的RNA标准品与未知样品一起运行，以验证任何RT-qPCR检测并确定误差程度。
