The presence of residual gold nanoparticles in samples interferes with the RT-qPCR assay used for gene expression profiling

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-017-0299-9) contains supplementary material, which is available to authorized users.

The use of HRM shifts in qPCR to investigate a much neglected aspect of interference by intracellular nanoparticles

“…A literature search narrowed the list of putative human reference genes down to 10 candidates before initiating any wet-bench experiments [ 6 ]. Thereafter, in silico analysis was performed in order to predict conformational changes under experimental conditions (see additional data in S1 File ).…”

“…A preliminary study was initially performed to test the primers’ ability to transcribe and amplify RNA from a human cell line using a SYBR Green-based RT-qPCR system [ 6 ]. The BEAS-2B cells were treated with 1 nM AuNPs for 24 h. After treatment, 1 μg of total RNA was extracted as previously reported [ 15 ].…”

“…Reference genes compensate for differences in the amount of starting material, efficiency of amplification or differences in transcription levels and expression between cells [ 4 , 5 ]. The identification of a stable reference gene for normalisation in engineered nanomaterial (ENM)-related RT-qPCR studies is essential [ 6 ]. Hence, the following candidates were selected: Human 18S ribosomal RNA (18S), beta-actin (Act-B), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta glucuronidase (GUS), Heat shock protein 90kDa alpha (cytosolic), class B (HSP90), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (cyclophilin A) (PPIA), succinate dehydrogenase complex subunit A flavoprotein (SDHA), TATA-box binding protein (TBP), and Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) [ 7 , 8 ].…”

Transcriptomics in Toxicogenomics, Part I: Experimental Design, Technologies, Publicly Available Data, and Regulatory Aspects

“…However, pristine MWCNTs did not cause the same level of interference, which emphasizes the role of surface chemistry in these types of analyses. Au-NPs were also reported to affect the reverse transcription efficiency in RT-qPCR [91]. Gao and colleagues later found that the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by some of the major types of metal oxide NPs (e.g., Fe2O3, ZnO, CeO2, FeO4, Al2O3, CuO, TiO2), but that this did not introduce mutations, i.e., the overall error rate was not significantly different and single nucleotide polymorphisms were not introduced [90].…”

Augmented Therapeutic Potential of Glutaminase Inhibitor CB839 in Glioblastoma Stem Cells Using Gold Nanoparticle Delivery

“…Contrary to commonly used assays such as MTT, Cell-Titer-Glo ® or Annexin A5, our focus lied further in the optimization of methods to assess the viability of the GSCs after treatment with Au-Polymer-CB839 NPs that do not rely on optical detection or addition of reagents. Au NPs may disturb assay readouts through their ability to absorb visible light, lumines, quench fluorescent signals or simply by interacting with assay components [ 39 , 40 , 41 ]. Thus, it is of high importance to have reliable and easy-to-apply methods to test the toxicity of drugs when using Au NPs.…”