Stability Assessment of Reference Genes for Reliable Analysis of Silver Nanoparticles Cytotoxicity in HepG2 Cell Line

Abstract

In spite of many reports on the toxicity of silver nanoparticles (AgNPs), the mechanisms underlying the toxicity are far from clear. For many years, evaluation of gene expression levels by reverse transcription real-time quantitative PCR (RT-qPCR) has been the discrete choice approach for discovering nanotoxicity-associated alterations and related mechanisms. The appropriate selection of reference genes is critical step for accurate data interpretation in RT-qPCR. In this study, five reference genes including GAPDH, YWHAZ, ACTB, TBP and 18s rRNA were evaluated to determine the most stable genes in a HepG2 cells under various AgNPs treatment conditions. Analysis by geNorm program ranked ACTB and YWHAZ in HepG2 cells as the most stable reference genes, While TBP was assessed as the most stable one by NormFinder, implying the importance of the employed algorithm in comparative interpretation of the data. 18s rRNA exhibited the least stable expression in this study using both algorithms. Cumulative data suggested the use of two reference genes (ACTB and TBP) for RT-qPCR data normalization under AgNPs treatment. Here, the selection of superior reference genes for RT-qPCR in HepG2 cells under different AgNPs treatment conditions was firstly investigated, and will be considered in future studies on mechanisms underlying nanosilver exposure.