title: Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria.
abstract: BACKGROUND
  Quantitative PCR is rapidly becoming the standard method for analyzing gene expression in a wide variety of biological samples however it can suffer from significant error if stably expressed reference genes are not identified on which to base the analysis. Suitable reference genes for qPCR experiments on Staphylococcus pseudintermedius have yet to be identified.
  
  
  RESULTS
  Three reference genes in S. pseudintermedius were identified and validated from a set of eight potential genes (proC, gyrB, rplD, rho, rpoA, ftsZ, recA, sodA). Two strains of S. pseudintermedius were used, and primer specificity and efficiency were confirmed and measured. Ranking of the genes with respect to expression stability revealed gyrB, rho and recA as the best reference genes. This combination was used to quantify expression of a single biofilm associated gene, icaA, in logarithmic, stationary and biofilm growth phases, revealing that expression was significantly upregulated in the biofilm growth phase in both strains.
  
  
  CONCLUSIONS
  Three reference genes, gyrB, rho and recA, were identified and validated for use as reference genes for quantitative PCR experiments in S. pseudintermedius. Also, the biofilm associated gene icaA was shown to be significantly upregulated in biofilm samples, consistent with its role in biofilm production.
authors.affiliation: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, 50 Stone Road, Ontario N1G 2 W1, Canada. ecrawf01@uoguelph.ca.
authors.forename: Evan C
authors.initials: EC
authors.lastname: Crawford
authors.forename: Ameet
authors.initials: A
authors.lastname: Singh
authors.forename: Devon
authors.initials: D
authors.lastname: Metcalf
authors.forename: Thomas W G
authors.initials: TW
authors.lastname: Gibson
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Weese
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D017931:DNA Primers / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D020043:Genes, Essential; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D013210:Staphylococcus / Q000235:genetics* / Q000254:growth & development
chemical_list: D017931:DNA Primers