title: Validation of the β-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis.
abstract: Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of β-amylase (β-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize β-amy1 analysis for study of the impact of β-amy1 expression upon barley end-use quality.
authors.affiliation: Department of Molecular Biology, Crop Research Institute, Prague, Czech Republic. ovesna@vurv.cz
authors.forename: Jaroslava
authors.initials: J
authors.lastname: Ovesná
authors.forename: Ladislav
authors.initials: L
authors.lastname: Kučera
authors.forename: Kateřina
authors.initials: K
authors.lastname: Vaculová
authors.forename: Kamila
authors.initials: K
authors.lastname: Štrymplová
authors.forename: Ilona
authors.initials: I
authors.lastname: Svobodová
authors.forename: Luigi
authors.initials: L
authors.lastname: Milella
mesh_terms: D015870:Gene Expression; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D001467:Hordeum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010940:Plant Proteins / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012639:Seeds / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D001614:beta-Amylase / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D001614:beta-Amylase