Yeast Reference Genes in Dynamic RT-qPCR Analysis Figure 4. Expression profile of MEP2 as a response to the relaxation from ammonium limitation. This figure represents the expression profile for MEP2 using different combinations of reference genes, which were selected from the candidate pool. The expression profiles were calculated using ACT1; a commonly selected reference gene, using FBA1 and ACT1; the two most stable genes identified from P-0 by geNorm, and using FBA1, TDH3, ACT1 and CCW12; the final set of most stable genes (A). The contribution of increasing the number of genes in the final reference set to the expression profile of MEP2 was investigated by adding CDC19 to the reference gene set (B). The effect of reducing number of genes in the final reference gene set on the expression profile of MEP2 was investigated. The number of reference genes included in the set was reduced by one each time starting from the least stable gene in the set (C). For each individual profile, the reference genes, which were used in the determination of MEP2 expression, were indicated by different colours in the legend. doi:10.1371/journal.pone.0038351.g004 Discussion Understanding the dynamic mechanism behind a biological process and the identification of the statistically meaningful changes in the expression levels of selected genes involved in the various processes require quantification and comparison of dynamic data. Real-time RT-qPCR is widely used to investigate the relative expression levels of target genes in detail. However, the selection of suitable reference genes imposes problems in the analysis of both transient and non-transient expression profiling studies. The quantification of the dynamic response by real-time RT-qPCR requires the identification of the reference genes, which display constant expression across all time points regardless of the different genetic or environmental perturbations. Although the commonly used reference genes previously reported in literature [4,5,6] constitute a suitable pool for the analysis of non-transient data, these genes were observed to fall short in fulfilling their roles as reference genes in the analysis of dynamic gene expression (data not shown). Therefore, a pool of candidate reference genes needs to be identified for dynamic studies. The most suitable set of reference genes would then be determined among the proposed candidates under the studied experimental conditions. In this study, high-throughput transient gene expression data were used to identify a set of candidate reference genes for real- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38351