during the inclusion of new dynamic datasets to be used for the determination of the candidate set and during the exclusion of the correlated gene pairs from the candidate pool while identifying the most stable genes. Materials and Methods Determination of the reference gene candidates In order to determine the reference candidates displaying stable expression throughout time course experiments, transcriptome data sets were used. Publicly available time series microarray data sets were downloaded from the Array Express database [12]. 31 different time-dependent experiments conducted with S. cerevisiae using Affymetrix Yeast Genome S98 and Yeast2 arrays (14 and 17 experiments, respectively) (Table S1) were first pre-processed with RMAExpress software applying quantile normalization [36]. For the multiple probe sets targeting the same gene, the average expression value would be used if there were no significant difference between the expression levels of the multiple probes and the larger value would be selected if there were a significant difference. The expression levels of the genes that were detected in both platforms were combined and a matrix of dimensions 54246888 was obtained for the 5424 identified transcripts measured at 888 time points in 31 different experiments. Assessment of expression stability by coefficient of variation approach The candidate reference genes with stable gene expression across time were determined by the coefficient of variation approach [2]. For each gene, the coefficient of variation (CV) of the expression value, which is defined as the ratio of the standard deviation to the mean was calculated and the CV values of the genes were then ranked. CV ~ s DmD CV values were determined for the combined data set of 31 different experiments and also individually for each experiment. The CV values were used in the assessment of stable expression profiles and the candidate gene list was determined. Composition of the combined datasets In order to determine the effect of inclusion of an additional dataset in the final cluster of experiments on the stability profile of the genes, combinations of 31 datasets were created. There were 465 possible combinations that contain 2 or 29 datasets and 31 possible combinations that contain 30 datasets. Stability profiles for each of these 496 combinations were identified. However, due to the excessive number of the possible combinations for the subsets containing 3 to 28 datasets, 1000 random combinations were generated for those subsets and the stability analysis was conducted for those random combinations. ni~ ci if civ1000 1000 if ciw1000 ni: number of generated subsets containing i datasets ci: number of all possible subsets containing i datasets where 1,i,31 Yeast Reference Genes in Dynamic RT-qPCR Analysis Confirmation of the stability of reference genes and their validation Strain and media. Homozygous hoD/hoD strain of S. cerevisiae from a genetic background of BY4743 (MATa/MATD his3D1/ his3D1 leu2D0/leu2D0 lys2D0/+ met15D0/+ ura3D0/ura3D0), pur- chased from the European Saccharomyces cerevisiae Archive for Functional Analysis [37], was kindly provided by Prof. Stephen G. Oliver. The absence of the deleted gene was verified using PCR- based methods [14]. The preculture was incubated in YPD medium (2% [wt/vol] D- glucose, 2% [wt/vol] peptone, 1% [wt/vol] yeast extract) at 30uC and 180 rpm in an orbital shaker. Synthetic defined medium was used in the fermentations [38]. Chemostat culture, experimental design and sample description. Cells were cultivated in B-Braun Biostat B plus fermenters with 1.5 L working volume of ammonium or glucose- limited synthetic defined medium. Ammonium or glucose was injected in an impulse-like manner into their respective limited cultures at steady state. The temperature was controlled at 30uC and the agitation was set to 800 rpm. The pH was controlled at 5.5 with 1 M NaOH and HCl. The dilution rate was set to 0.1 hr21. The air flow into the fermenters was adjusted at 1.5 L/ min to keep dissolve oxygen saturation above 80% throughout the experiment. A total of 14 time-dependent RNA samples were collected at steady state, in the first minute with 20 second intervals, with 8 minute intervals for 32 minutes and then hourly for 5 hours following the impulse. The sample volume was 5 ml of culture at an OD range of 1.2–1.4. The samples were immediately frozen in liquid nitrogen and stored at 280uC until further processing. Nucleic acid extraction. RNA was isolated from samples with the Qiagen RNeasy mini kit (Cat no: 74106) using the ‘‘RNeasy protocol for extracting yeast via enzymatic lysis’’ using robotic workstation, QIAcube (Qiagen, USA), modified by the manufacturer for yeast applications. b-mercaptoethanol (Cat no: 444203) was purchased from Merck. The nucleic acid concentra- tions were determined and the purity of the RNA (A260/A280) was confirmed using NanoDrop (ND-1000, Thermo Scientific) (Table S7). The observed yield was ensured to be at least 90% of the expected yield. RNA integrity was determined using Bioanalyzer 2100 (Agilent Technologies, USA) using RNA6000 Nanokit (Agilent Technologies, USA). RIN numbers and the electropho- resis traces were provided in Text S1. The absence of any DNA contamination was confirmed using RNase treated samples as negative control. Reverse transcription. In order to unify the initial concen- tration of RNA in the analyses, all samples were diluted to the same concentration prior to the real-time RT-qPCR application. Reverse transcription was carried out at 50uC for 30 minutes. The reaction was allowed to proceed with 80 ng/ml in a reaction volume of 12.5 ml. QuantiTect RT mix (Qiagen, USA, Cat no: 204245) was used at a ratio of 0.01 total reaction volume. Assays were conducted in triplicates. The synthesized cDNA template was immediately allowed to proceed with the polymerase chain reaction. qPCR target information. The PCR product (amplicon) was determined to be either 100–150 base pairs long or 200–250 base pairs long. The gene symbols, sequence accession numbers, location of the amplicon, amplicon lengths, in silico PCR results for specificity is provided in Text S2. qPCR oligonucleotides. The primer length was selected to be in the range of 18–24 nucleotides. The GC content of the primers ranged between 50 to 60 per cent. The melting temperatures of the primers were in the range of 55–58uC. PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e38351