Yeast Reference Genes in Dynamic RT-qPCR Analysis Figure 2. Expression profile of HAP4 in response to the relaxation from glucose limitation. This figure represents the expression profile for HAP4 using different combinations of reference genes, which were selected from the candidate pool. The expression profiles were calculated using TDH3; a commonly selected reference gene, using ARF1 and CDC19; the two most stable genes identified from P-0 by geNorm, and using TPI1, FBA1, CDC19 and ACT1; 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 HAP4 was investigated by adding ARF1 and GCN4 to the reference gene set in the given order (B). The effect of reducing number of genes in the final reference gene set on the expression profile of HAP4 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 HAP4 expression, were indicated by different colours in the legend. doi:10.1371/journal.pone.0038351.g002 pool consisting of at most four reference genes was determined to be sufficient. The change in the gene expression profile within the first minute upon inclusion of ARF1 in the reference gene set was similar to the response that was observed when the ARF1-CDC19 gene pair was used as reference. The common denominator in the ARF1-CDC19 reference gene set and the TPI1-FBA1-CDC19-ACT1 reference gene set was CDC19, which might indicate that the general trend in HAP4 expression profile was observed owing to the presence of this gene. The unexpected increase in the gene expression at the 20th second post impulse was observed due to the inclusion of ARF1 in the reference gene set (Figure 2-a, 2-b). The observation that the inclusion of the more genes in the reference set did not provide any improvement led to the thinking that the reference gene set would be further reduced in size. For this purpose, the number of reference genes included in the set was reduced by one each time starting from the least stable gene in the set. The exclusion of ACT1 from the reference gene set resulted in an unexpected increase at the 20th second post impulse. A further exclusion of CDC19 from the set caused an even steeper increase in HAP4 expression at the 20th second post impulse in addition to PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38351