title: Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus.
abstract: Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis.
authors: Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium.
authors: Jacob
authors: J
authors: Pollier
authors: Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium.
authors: Robin
authors: R
authors: Vanden Bossche
authors: VTT Technical Research Centre of Finland, P.O. Box 1000, 02044-VTT, Finland.
authors: Heiko
authors: H
authors: Rischer
authors: Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium. Electronic address: alain.goossens@psb.vib-ugent.be.
authors: Alain
authors: A
authors: Goossens
mesh_terms: D030008:Catharanthus / Q000235:genetics*; D020869:Gene Expression Profiling; D017343:Genes, Plant; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger
keywords: BestKeeper; Catharanthus roseus; NormFinder; Reference gene; Transcript normalization; geNorm; qPCR