title: Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions.
abstract: BACKGROUND
  Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species.
  
  
  RESULTS
  Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene.
  
  
  CONCLUSIONS
  Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.
authors: Section for Microbiology, Immunology and Parasitology, Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, PO Box 8146 Dep, Oslo 0033, Norway.
authors: Espen
authors: E
authors: Brudal
authors: Hanne Cecilie
authors: HC
authors: Winther-Larsen
authors: Duncan John
authors: DJ
authors: Colquhoun
authors: Samuel
authors: S
authors: Duodu
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005399:Fishes / Q000382:microbiology*; D005603:Francisella / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D005798:Genes, Bacterial; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers