Abstract
Genetic molecular studies used to understand potential risks of engineered nanomaterials (ENMs) are incomplete. Intracellular residual ENMs present in biological samples may cause assay interference. This report applies the high-resolution melt (HRM) feature of RT-qPCR to detect shifts caused by the presence of gold nanoparticles (AuNPs). A universal RNA standard (untreated control) sample was spiked with known amounts of AuNPs and reverse transcribed, where 10 reference genes were amplified. The amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves were analysed and detected interference caused by AuNPs, which differed according to the amount of AuNP present (i.e. semi-quantitative). Whether or not the assay interference was specific to the reverse transcription or the PCR amplification step was tested. The study was extended to a target gene-of-interest (GOI), Caspase 7. Also, the effect on in vitro cellular samples was assessed (for reference genes and Caspase 7). This method can screen for the presence of AuNPs in RNA samples, which were isolated from biological material in contact with the nanomaterials, i.e., during exposure and risk assessment studies. This is an important quality control procedure to be implemented when quantifying the expression of a GOI from samples that have been in contact with various ENMs. It is recommended to further examine 18S, PPIA and TBP since these were the most reliable for detecting shifts in the difference curves, irrespective of the source of the RNA, or, the point at which the different AuNPs interacted with the assay.
Highlights
Gene expression studies commonly use Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) due to the sensitivity of the technique, as well as, the high diversity where different target genes-of-interest (GOI) can be studied
After testing of the universal RNA standard, the experiment was repeated with RNA that was obtained from BEAS-2B cells that had been treated with 1 nM PCOOH amounts of citrate-stabilised gold nanoparticles (AuNPs) for 0.5 h, 1 h, 2 h and 24 h
Any differences observed were due to the AuNPs, which appeared to influence the transcription of RNA into cDNA to a higher degree than the PCR amplification efficiency
Summary
Gene expression studies commonly use Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) due to the sensitivity of the technique, as well as, the high diversity where different target genes-of-interest (GOI) can be studied. It relies on the normalisation of the expression data between samples, e.g. use of reference genes as internal controls [1,2,3]. 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]. HRM shifts in qPCR to investigate interference by intracellular nanoparticles
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