title: A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis.
abstract: The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a 2(-ΔΔC(q)) (comparative C(q) method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.
authors.affiliation: Interdepartmental Research Group on Metabolic Engineering, Federal University of Pernambuco, Av. Moraes Rego 1235, Recife, PE 50670-901, Brazil. will.barros.pita@gmail.com
authors.forename: Will
authors.initials: W
authors.lastname: de Barros Pita
authors.forename: Fernanda Cristina Bezerra
authors.initials: FC
authors.lastname: Leite
authors.forename: Anna Theresa
authors.initials: AT
authors.lastname: de Souza Liberal
authors.forename: Luciana Filgueira
authors.initials: LF
authors.lastname: Pereira
authors.forename: Marcelo Falsarella
authors.initials: MF
authors.lastname: Carazzolle
authors.forename: Gonçalo Amarante
authors.initials: GA
authors.lastname: Pereira
authors.forename: Marcos Antonio
authors.initials: MA
authors.lastname: de Morais
mesh_terms: D017931:DNA Primers; D055167:Dekkera / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger