title: lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.
abstract: BACKGROUND
  Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions.
  
  
  RESULTS
  We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions.
  
  
  CONCLUSIONS
  We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.
authors.affiliation: Unité Microbiologie, Adaptation, Pathogénie CNRS-INSA-UCBL UMR 5240, Université Lyon 1, Villeurbanne, France. hommais@univ-lyon1.fr
authors.forename: Florence
authors.initials: F
authors.lastname: Hommais
authors.forename: Ouafa
authors.initials: O
authors.lastname: Zghidi-Abouzid
authors.forename: Christine
authors.initials: C
authors.lastname: Oger-Desfeux
authors.forename: Emilie
authors.initials: E
authors.lastname: Pineau-Chapelle
authors.forename: Frederique
authors.initials: F
authors.lastname: Van Gijsegem
authors.forename: William
authors.initials: W
authors.lastname: Nasser
authors.forename: Sylvie
authors.initials: S
authors.lastname: Reverchon
mesh_terms: D017360:Arabidopsis / Q000382:microbiology*; D030541:Databases, Genetic; D004755:Enterobacteriaceae / Q000235:genetics*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D044043:Pectobacterium / Q000235:genetics / Q000254:growth & development; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012689:Sequence Homology, Nucleic Acid; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D012329:RNA, Bacterial