title: Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization.
abstract: Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.
authors.affiliation: Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy. Electronic address: verena.stenico2@unibo.it.
authors.forename: Verena
authors.initials: V
authors.lastname: Stenico
authors.affiliation: Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Baffoni
authors.affiliation: Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gaggìa
authors.affiliation: Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Biavati
mesh_terms: D000328:Adult; D001644:Bifidobacterium / Q000187:drug effects / Q000235:genetics*; D001646:Bile / Q000378:metabolism; D019295:Computational Biology; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D005798:Genes, Bacterial; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D012984:Software
keywords: BestKeeper; Bifidobacterium adolescentis; NormFinder; Reference genes; qRT-PCR; sdhA