Brudal Espen, Winther-Larsen Hanne Cecilie, Colquhoun Duncan John, Duodu Samuel
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.
BMC Res Notes. 2013 Mar 2;6:76. doi: 10.1186/1756-0500-6-76.
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.
逆转录定量聚合酶链反应已成为监测许多细菌物种中mRNA转录以响应不同环境条件的一项强大技术。然而，要正确评估数据，需要准确可靠地使用在实验过程中转录不变的参考基因。在本研究中，通过暴露于不同生长条件来验证8个候选参考基因在诺氏弗朗西斯菌两个亚种的三株菌株中的转录稳定性，诺氏弗朗西斯菌是一种可在冷水和暖水鱼类中致病的病原体。
在指数生长期、稳定期和铁限制生长条件下，对编码DNA促旋酶（gyrA）、RNA聚合酶β亚基（rpoB）、DNA聚合酶I（polA）、细胞分裂蛋白（ftsZ）、外膜蛋白（fopA）、核黄素生物合成蛋白（ribC）、16S核糖体RNA（16S rRNA）和DNA解旋酶（uvrD）的基因的相对转录水平进行了定量。使用与毒力相关的细胞内生长位点亚基C（iglC）基因测试了所选参考基因对可靠解释基因表达数据的适用性。
尽管在所研究的三株菌株中参考基因的转录稳定性略有不同，但fopA、ftsZ和polA被证明是最稳定的，适用于诺氏弗朗西斯菌亚种中基因转录的标准化。
