Validation of Reference Genes for Silver Nanotoxicity Analysis in MCF-7 Cell Line

Abstract

Following the increasing use of silver nanoparticles (AgNPs) and the growing exposure to these particles, nanotoxicity has become a general concern among researchers around the world. Although quantitative real time PCR (RT-qPCR) is the technique of choice in genotoxicity assessment, use of unconfirmed reference genes could result in data misinterpretation. GeNorm and NormFinder algorithms assist in the selection of appropriate reference genes in RT-qPCR expression analysis. Using these two algorithms, here, the stability of six reference genes’ expression including GAPDH, ACTB, UBC, TBP, HPRT-1 and YWHAZ was evaluated in MCF-7 cells treated with different concentrations (under IC50 estimated by MTT assay) of AgNP. results showed that cytotoxicity can occur in a dose dependent fashion in MCF-7 cells with IC50 value of 5.495 µg/ml. Cumulative analysis by geNorm and NormFinder programs indicated that the normalization of the RT-qPCR data under AgNPs treatment in MCF-7 cells would be greatly improved utilizing two reference genes including TBP and GAPDH. This study is the first investigation for the selection of suitable reference genes for RT-qPCR analysis in MCF-7 cells treated with different concentration of AgNPs, and could be used in future researches established to reveal mechanisms underlying nanosilver exposure.