Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Abstract

Background: Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.

Yeast Tolerance to Various Stresses Relies on the Trehalose-6P Synthase (Tps1) Protein, Not on Trehalose

“…qPCR Assays Quality Control and Data Analysis-Most of the primer sets for qPCR, especially the reference genes, were previously described and validated (30). New pairs of primers were designed using Vector NTI advance v11 (Life Technologies, Inc.).…”

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

“…To confirm that Upc2 is involved in the regulation of gene expression of ergosterol biosynthetic enzymes, we performed quantitative gene expression analysis of ERG11 by real-time PCR using the upc2/ecm22-knockout strains with wild-type and mutant UPC2 alleles. We measured the mRNA levels of ERG11 genes that were normalized by TAF10 gene as an internal control 33 . The data indicate that the mRNA expression levels of ERG11 correlate with the promotor activities of ERG2 and ergosterol levels as expected ( Supplementary Fig.…”

A sulphite-inducible form of the sulphite efflux gene SSU1 in a Saccharomyces cerevisiae wine yeast

“…Raw threshold cycle data were processed using the geNorm online tool (http://medgen.ugent.be/ jvdesomp/genorm/) (Vandesompele et al, 2002). This allowed identification of the most stable genes in the dataset (data not shown) and the optimal number of housekeeping genes for normalization, taking into account current constraints for real-time PCR data (Bustin et al, 2005), also recently described for yeasts (Teste et al, 2009). Actin (ACT1) and translational elongation factor EF-1a (TEF1) were chosen as reference genes; all data were normalized to both ACT1 and TEF1 simultaneously, using the iQ5 software provided by Bio-Rad (Vandesompele et al, 2002).…”