Authors: Natasha M. Sanabria And Mary Gulumian
Source: Sanabria And Gulumian ?J Nanobiotechnol (2017) 15:72
Background: 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.
Results: 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.
Conclusions: 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.
Keywords: RT-qPCR, AuNPS, Nanoparticles, Engineered nanomaterials, Toxicology, Risk assessment