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Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses.
Dominika Borowska1, Richard Kuo1, Richard A Bailey2
1The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, Scotland, United Kingdom.
Plos One
|December 4, 2019
Summary
We developed a high-throughput qPCR assay to analyze chicken immune gene expression. Blood cells proved reliable indicators of immune responses in commercial chickens under different biosecurity conditions.
Area of Science:
- Immunology
- Avian Biology
- Molecular Biology
Background:
- Assessing avian immune responses requires sensitive, high-throughput methods.
- Existing methods may lack the capacity for simultaneous analysis of numerous immune genes.
Purpose of the Study:
- To develop and validate a highly multiplexed quantitative polymerase chain reaction (qPCR) assay for analyzing chicken immune gene transcription.
- To evaluate the utility of this assay in commercial broiler chickens under varying biosecurity levels.
Main Methods:
- A 96.96 Fluidigm Dynamic Array was utilized for high-throughput gene expression analysis.
- A panel of 89 immune-related genes was selected based on RNA-seq and published data.
- The array was applied to RNA from various tissues (gut-associated lymphoid tissues, bursa, spleen, peripheral blood leukocytes) of broiler chickens.
Main Results:
- The multiplex qPCR assay demonstrated high specificity and optimized performance.
- Blood cells showed the highest number of significantly differentially transcribed immune genes between birds under different biosecurity levels.
- Results were validated using conventional qPCR for selected genes.
Conclusions:
- A validated, highly multiplexed qPCR platform is available for evaluating chicken immune responses.
- This platform has applications in selective breeding, pathogen-host interaction studies, vaccine screening, and predicting immune robustness.
