Pharmacogenomics, toxicogenomics, and small RNA expression analysis are three of 
the most active research topics in the biological, biomedical, pharmaceutical, 
and toxicological fields. All of these studies are based on gene expression 
analysis, which requires reference genes to reduce the variations derived from 
different amounts of starting materials and different efficiencies of RNA 
extraction and cDNA synthesis. Thus, accurate normalization to one or several 
constitutively expressed reference genes is a prerequisite to valid gene 
expression studies. Although selection of reliable reference genes has been 
conducted in previous studies in several animals and plants, no research has 
been focused on pharmacological targets, and very few studies have had a 
toxicological context. More interestingly, no studies have been performed to 
identify reference genes for small RNA analysis although small RNA, particularly 
microRNA (miRNA)-related research is currently one of the fastest-moving topics. 
In this study, using MCF-7 breast cancer cells as a model, we employed 
quantitative real-time PCR (qRT-PCR), one of the most reliable methods for gene 
expression analysis in many research fields, to evaluate and to determine the 
most reliable reference genes for pharmacogenomics and toxicogenomics studies as 
well as for small RNA expression analysis. We tested the transcriptional 
expression of five protein-coding genes as well as five non-coding genes in 
MCF-7 cells treated with five different pharmaceuticals or toxicants [paclitaxel 
(PTX), gossypol (GOS), methyl jasmonate (JAS), L-nicotine (NIC), and melamine 
(mela)] and analyzed the stability of the selected reference genes by four 
different methods: geNorm, NormFinder, BestKeeper, and the comparative ΔCt 
method. According to our analysis, a protein-coding gene, hTBCA and four 
non-coding genes, hRNU44, hRNU48, hU6, and hRNU47, appear to be the most 
reliable reference genes for the five chemical treatments. Similar results were 
also obtained in dose-response and time-course assays with gossypol (GOS) 
treatment. Our results demonstrated that traditionally used reference genes, 
such as 18s RNA, β-actin, and GAPDH, are not reliable reference genes for 
pharmacogenomics and toxicogenomics studies. In contrast, hTBCA and small RNAs 
are more stable during drug treatment, and they are better reference genes for 
pharmacogenomics and toxicogenomics studies. To widely use these genes as 
reference genes, these results should be corroborated by studies with other 
human cell lines and additional drugs classes and hormonal treatments.