Abstract
BackgroundRT-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.ResultsAmplification 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.ConclusionsAuNPs 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.
Highlights
reverse transcription quantitative polymerase chain reaction (RT-qPCR) is routinely used in expression profiling of toxicity pathway genes
This work, promotes awareness regarding the effects of residual intracellular engineered nanomaterials (ENMs)
Thereafter, a preliminary test was performed using SYBR Green in order to screen these primers. This part of the study was completed by analysing the RNA obtained from primary and immortalized human bronchial epithelial cells (BEAS-2B) cells that had been treated with gold nanoparticles (AuNPs)
Summary
RT-qPCR is routinely used in expression profiling of toxicity pathway genes. 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. The risk of exposure has increased and the possible toxicity of these ENMs must be determined. Gene expression assays that rely heavily on RNA with excellent quality should be undertaken with great care, since it has already been shown that AuNPs could interfere with traditional RNA analyses [1]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
