Selection of reference genes for quantitative real-time PCR analysis in chicken ovary following silver nanoparticle treatment

Abstract

Reliable results of quantitative real time PCR (qPCR) analysis require normalization of target gene expression level using reference genes (RGs). However housekeeping genes expression may vary under experimental conditions, so selection of the proper RGs is a crucial step in a qPCR analysis. Several algorithms have been developed to address this problem: geNorm, NormFinder and BestKeeper. In this study, we have used these three tools to evaluate the stability of RGs in the ovarian tissues of hens treated with silver nanoparticles. Eight genes were selected for the validation: HPRT, HMBS, VIM, SDHA, TBP, RPL13, GAPDH and 18S rRNA. According to geNorm the best combination of reference genes is SDHA and TPP. NormFinder also selected SDHA as the most suitable gene, but in combination with RPL13. Analysis in BestKeeper showed that SDHA, RPL13 might be the best choice in gene expression studies using the chicken ovary. In conclusion, the results obtained depend on the algorithm used and it arises from the diverse calculation strategies used in these programs. The outcome from the NormFinder is considered to be the most trustworthy and used in further qPCR analysis.