unexpected increases in gene expression in the 60th second and another slight increase at the 24th minute. Utilization of TPI1 as the sole reference gene for the normalization of HAP4 expression yielded a similar expression profile for this gene to that of using the geometric average of the Ct values for TPI1, FBA1 and CDC19 (Figure 2-c). The increase in the expression of HAP4 at the 20th second could only be prevented via the inclusion of ACT1 in the reference gene set raised a question of whether this gene could be used as a single reference gene in determining the expression profiles under the stated conditions. Moreover, ACT1 was frequently reported as a reference gene in yeast gene expression studies via real-time RT-qPCR [6]. However, the use of ACT1 as the sole reference gene did not yield a decreasing expression profile for HAP4. The similarity between the expression profiles when TPI1 was used alone or in conjunction with FBA1 and CDC19 aroused curiosity regarding whether using the geometric average of the Ct values for FBA1 and CDC19 alone would result in an adequate expression profile for HAP4. However this reference gene pair caused a steep increase in HAP4 expression at the 20th second post impulse (Figure S7). These results indicated that the reference gene set comprised of TPI1, FBA1, CDC19, ACT1 was suitable for determining gene expression in response to an impulse-like perturbation in glucose levels. Case Study II: Selection and validation of the most stable reference genes during ammonium perturbation In order to identify correlated gene pairs from the candidate list, Pearson correlation coefficients between expression values of each pair of the candidates were calculated. Analysis of the PCCs between the gene pairs among the candidate gene set revealed the presence of one gene pair with a PCC greater than 0.85 in response to relaxation of ammonium limitation (Table S5). TDH3 was correlated with PDC1. Three different pools of candidate genes were formed in order to investigate the effect of the inclusion of correlated gene pairs in the reference gene set. P-0 was the pool including all candidate genes. P-1 and P-2 were created by excluding TDH3 and PDC1 from the candidate list, respectively. One other alternative pool was constructed by excluding both of these correlated genes from the candidate list (P-3). The eleven candidates were ranked based on the stability of their expression profiles using NormFinder and geNorm as it was the case for the glucose perturbation. Additionally, geNorm algorithm was also used to determine the stability ranking of genes in the pools, in which only the genes with a correlation of ,D0:85D among their expression profiles, were left (P-1 to P-3). The genes were scored such that the average stability rankings were obtained using NormFinder and geNorm for each pool separately. The total score for each candidate gene was calculated by summing up its individual scores for each case; P-0 to P-3. The genes were then ranked based on their total scores from the minimum score to the maximum score indicating an order from the most stable to the least stable gene. Although the minimum number of reference genes to be used could be selected as 3 in order to make sure that at least one stable gene remained even after the removal of the correlated gene pair in P-3, the number of reference genes was selected as 4 for the sake of consistency with Case Study I (Figure S8). Indeed the analysis conducted by geNorm also demonstrated that 4 genes should be used as reference under the stated conditions (Figure S9). This ranking indicated that the lowest total scores were obtained for FBA1, TDH3, ACT1 and CCW12. In this case, geNorm did not capture the gene pair with the highest correlation as the most stable pair. However, although the most stable gene was identified as FBA1 and the 4 least stable genes were identified as GCN4, ARF1, PDC1 and RPS26A by both Yeast Reference Genes in Dynamic RT-qPCR Analysis geNorm and NormFinder, the stability orders for the remaining of the set were considerably different by either one of the algorithms. Removal of the correlation among the candidate genes did not change the stability rankings determined by geNorm such that both algorithms would yield similar stability profiles. If the three candidate pools (P-1 to P-3) were analyzed, FBA1 and ACT1 would fall into the stable gene set as it was the case that was determined by NormFinder (Figure 3, Figure S8, Figure S10). FBA1, TDH3, CCW12 and ACT1 were determined as the four most stable genes among all candidates as indicated by their scores (Figure 3). A set of four reference genes; FBA1, TDH3, CCW12 and ACT1 were determined to constitute a suitable pool for the analysis of gene expression in response to an impulse-like ammonium perturbation. The change in the expression level of MEP2, an ammonium transporter, was investigated in order to test the performance of this reference gene pool. MEP2, whose mRNA accumulation was more abundant in cells grown in limiting concentrations of ammonia than in high concentrations [18] was monitored in response to an impulse-like addition of ammonium into its limited culture. Therefore it could be expected that MEP2 would display a decreasing expression profile in response to a sudden increase in the amount of available ammonium in the medium. In order to evaluate the importance of determining a suitable reference gene set, which would be used in the normalization of the expression profile of the gene of interest, three different sets of reference genes were used to calculate the relative expression profile of MEP2. Initially ACT1; one of the commonly used reference genes was used as the sole gene for normalization. Then, the geometric average of the Ct values for FBA1-ACT1; the gene pair, which were identified as the most stable pair in the analysis conducted by geNorm using P-0 was used as the reference gene set for the normalization of MEP2 expression. In the third case, the geometric average of the Ct values of FBA1, TDH3, CCW12 and Figure 3. Scoring of the candidate genes in Case Study II. The cells representing the scores of the genes in different pools were colour coded. A coding scale from red to green indicated decreasing stability. The score of the genes in each pool was indicated by the number assigned to that cell in a quantitative manner; the smallest score indicating the most stable gene and the largest score indicating the least stable one. The stability of the gene associated with that score was also indicated by the colour assigned to that cell in a qualitative manner. The scoring of the four different pools of candidate genes was determined as the average ranking for each gene as indicated by the NormFinder and the geNorm analyses and the results were provided in the first six columns. The analysis was carried out using all genes (P-0), and using the 3 pools, each excluding a different set of correlated genes (P-1 to P-3). The total score for each gene was provided in the last column by summing up all of its scores through P-0 to P-3. doi:10.1371/journal.pone.0038351.g003 PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38351