title: Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data.
abstract: Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis.
authors.affiliation: Institute of Health Sciences, Federal University of Bahia, Salvador, BA, Brazil.
authors.forename: Daiane M
authors.initials: DM
authors.lastname: Carvalho
authors.forename: Pablo H
authors.initials: PH
authors.lastname: de Sá
authors.forename: Thiago L P
authors.initials: TL
authors.lastname: Castro
authors.forename: Rodrigo D
authors.initials: RD
authors.lastname: Carvalho
authors.forename: Anne
authors.initials: A
authors.lastname: Pinto
authors.forename: Danilo J P
authors.initials: DJ
authors.lastname: Gil
authors.forename: Priscilla
authors.initials: P
authors.lastname: Bagano
authors.forename: Bruno
authors.initials: B
authors.lastname: Bastos
authors.forename: Lilia F M
authors.initials: LF
authors.lastname: Costa
authors.forename: Roberto
authors.initials: R
authors.lastname: Meyer
authors.forename: Artur
authors.initials: A
authors.lastname: Silva
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Rommel T J
authors.initials: RT
authors.lastname: Ramos
authors.forename: Luis G C
authors.initials: LG
authors.lastname: Pacheco
mesh_terms: D016925:Corynebacterium pseudotuberculosis / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D005798:Genes, Bacterial; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D017423:Sequence Analysis, RNA