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Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

Sarah Van Lent (UGent) and Daisy Vanrompay (UGent)
(2016) JOURNAL OF VETERINARY RESEARCH. 60(4). p.403-409
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Abstract
Introduction: Chiamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chiamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci CallO was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.
Keywords
birds, Chiamydia psittaci, RT-qPCR, normalisation, references genes, REVERSE-TRANSCRIPTION PCR, RT-QPCR, STAPHYLOCOCCUS-AUREUS, LIFE-CYCLE, TRACHOMATIS, STRAINS, INFECTION, TROPISM, MODELS, L2

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MLA
Van Lent, Sarah, and Daisy Vanrompay. “Chlamydia Psittaci Reference Genes for Normalisation of Expression Data Differ Depending on the Culture Conditions and Selected Time Points During the Chlamydial Replication Cycle.” JOURNAL OF VETERINARY RESEARCH 60.4 (2016): 403–409. Print.
APA
Van Lent, S., & Vanrompay, D. (2016). Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle. JOURNAL OF VETERINARY RESEARCH, 60(4), 403–409.
Chicago author-date
Van Lent, Sarah, and Daisy Vanrompay. 2016. “Chlamydia Psittaci Reference Genes for Normalisation of Expression Data Differ Depending on the Culture Conditions and Selected Time Points During the Chlamydial Replication Cycle.” Journal of Veterinary Research 60 (4): 403–409.
Chicago author-date (all authors)
Van Lent, Sarah, and Daisy Vanrompay. 2016. “Chlamydia Psittaci Reference Genes for Normalisation of Expression Data Differ Depending on the Culture Conditions and Selected Time Points During the Chlamydial Replication Cycle.” Journal of Veterinary Research 60 (4): 403–409.
Vancouver
1.
Van Lent S, Vanrompay D. Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle. JOURNAL OF VETERINARY RESEARCH. 2016;60(4):403–9.
IEEE
[1]
S. Van Lent and D. Vanrompay, “Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle,” JOURNAL OF VETERINARY RESEARCH, vol. 60, no. 4, pp. 403–409, 2016.
@article{8502010,
  abstract     = {Introduction: Chiamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chiamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases.
Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci CallO was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm.
Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle.
Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.},
  author       = {Van Lent, Sarah and Vanrompay, Daisy},
  issn         = {2450-7393},
  journal      = {JOURNAL OF VETERINARY RESEARCH},
  keywords     = {birds,Chiamydia psittaci,RT-qPCR,normalisation,references genes,REVERSE-TRANSCRIPTION PCR,RT-QPCR,STAPHYLOCOCCUS-AUREUS,LIFE-CYCLE,TRACHOMATIS,STRAINS,INFECTION,TROPISM,MODELS,L2},
  number       = {4},
  pages        = {403--409},
  title        = {Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle},
  url          = {http://dx.doi.org/10.1515/jvetres-2016-0060},
  volume       = {60},
  year         = {2016},
}

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