Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis inEdwardsiella tarda.
Edwardsiella tardais a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors ofE. tarda. Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene inE. tarda, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (recA,uup,rpoB,rho,topA,gyrA,groEL,rpoD, and16S rRNA) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that16S rRNAwas more stable than the other genes at different culture growth phases. However, at the same culture time,topAwas identified as the reference gene under the conditions of different strains, different culture media, and infection, whereasgyrAwas identified under the condition of different temperatures. Thus, in experiments, the expression ofgapAandfbaAinE. tardawas analyzed by RT-qPCR using16S rRNA,recA, anduupas the reference genes. The results showed that16S rRNAwas the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
Zhongyang
Z
Sun
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
Jia
J
Deng
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.|Shanghai Engineering Research Center of Marine Cultured Animal Vaccines, Shanghai 200237, P.R. China.
Haizhen
H
Wu
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.|Shanghai Engineering Research Center of Marine Cultured Animal Vaccines, Shanghai 200237, P.R. China.
Qiyao
Q
Wang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.|Shanghai Engineering Research Center of Marine Cultured Animal Vaccines, Shanghai 200237, P.R. China.|Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai 200237, P.R. China.
Yuanxing
Y
Zhang
Edwardsiella tarda; RT-qPCR; Reference gene; culture condition