Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions

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 cha...

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Bibliographic Details
Published in:BMC research notes 2013-03, Vol.6 (1), p.76-76, Article 76
Main Authors: Brudal, Espen, Winther-Larsen, Hanne Cecilie, Colquhoun, Duncan John, Duodu, Samuel
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Cell division
DNA Primers
Fishes - microbiology
Francisella
Francisella - genetics
Francisella - growth & development
Francisella - pathogenicity
Gene expression
Genes, Bacterial
Microbiology
Proteins
Reverse Transcriptase Polymerase Chain Reaction - methods
Studies
Transcription, Genetic
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Summary: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. 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. 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.
ISSN:1756-0500
1756-0500
DOI:10.1186/1756-0500-6-76