A novel strategy for selection and validation of reference genes in dynamic multidimensional experimental design in yeast.

BACKGROUND: Understanding the dynamic mechanism behind the transcriptional organization of genes in response to varying environmental conditions requires time-dependent data. The dynamic transcriptional response obtained by real-time RT-qPCR experiments could only be correctly interpreted if suitable reference genes are used in the analysis. The lack of available studies on the identification of candidate reference genes in dynamic gene expression studies necessitates the identification and the verification of a suitable gene set for the analysis of transient gene expression response. PRINCIPAL FINDINGS: In this study, a candidate reference gene set for RT-qPCR analysis of dynamic transcriptional changes in Saccharomyces cerevisiae was determined using 31 different publicly available time series transcriptome datasets. Ten of the twelve candidates (TPI1, FBA1, CCW12, CDC19, ADH1, PGK1, GCN4, PDC1, RPS26A and ARF1) we identified were not previously reported as potential reference genes. Our method also identified the commonly used reference genes ACT1 and TDH3. The most stable reference genes from this pool were determined as TPI1, FBA1, CDC19 and ACT1 in response to a perturbation in the amount of available glucose and as FBA1, TDH3, CCW12 and ACT1 in response to a perturbation in the amount of available ammonium. The use of these newly proposed gene sets outperformed the use of common reference genes in the determination of dynamic transcriptional response of the target genes, HAP4 and MEP2, in response to relaxation from glucose and ammonium limitations, respectively. CONCLUSIONS: A candidate reference gene set to be used in dynamic real-time RT-qPCR expression profiling in yeast was proposed for the first time in the present study. Suitable pools of stable reference genes to be used under different experimental conditions could be selected from this candidate set in order to successfully determine the expression profiles for the genes of interest.

How yeast re-programmes its transcriptional profile in response to different nutrient impulses.

BACKGROUND: A microorganism is able to adapt to changes in its physicochemical or nutritional environment and this is crucial for its survival. The yeast, Saccharomyces cerevisiae, has developed mechanisms to respond to such environmental changes in a rapid and effective manner; such responses may demand a widespread re-programming of gene activity. The dynamics of the re-organization of the cellular activities of S. cerevisiae in response to the sudden and transient removal of either carbon or nitrogen limitation has been studied by following both the short- and long-term changes in yeast's transcriptomic profiles. RESULTS: The study, which spans timescales from seconds to hours, has revealed the hierarchy of metabolic and genetic regulatory switches that allow yeast to adapt to, and recover from, a pulse of a previously limiting nutrient. At the transcriptome level, a glucose impulse evoked significant changes in the expression of genes concerned with glycolysis, carboxylic acid metabolism, oxidative phosphorylation, and nucleic acid and sulphur metabolism. In ammonium-limited cultures, an ammonium impulse resulted in the significant changes in the expression of genes involved in nitrogen metabolism and ion transport. Although both perturbations evoked significant changes in the expression of genes involved in the machinery and process of protein synthesis, the transcriptomic response was delayed and less complex in the case of an ammonium impulse. Analysis of the regulatory events by two different system-level, network-based approaches provided further information about dynamic organization of yeast cells as a response to a nutritional change. CONCLUSIONS: The study provided important information on the temporal organization of transcriptomic organization and underlying regulatory events as a response to both carbon and nitrogen impulse. It has also revealed the importance of a long-term dynamic analysis of the response to the relaxation of a nutritional limitation to understand the molecular basis of the cells' dynamic behaviour.