Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses

Autor: Richard Kuo, Mark P. Stevens, Richard A. Bailey, Dominika Borowska, Lonneke Vervelde, Peter K. Kaiser, Kellie A. Watson
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0301 basic medicine
Avian immune system
Molecular biology
Physiology
Assay
Gene Expression
Artificial Gene Amplification and Extension
Breeding
Polymerase Chain Reaction
Poultry
law.invention
0403 veterinary science
Transcriptome
Sequencing techniques
law
Immune Physiology
Gene expression
High throughput
Medicine and Health Sciences
Leukocytes
Multiplex
Gamefowl
RNA-Seq
Immune Response
Polymerase chain reaction
Vaccines
Multidisciplinary
Eukaryota
RNA sequencing
04 agricultural and veterinary sciences
Microfluidic Analytical Techniques
Chicken
qPCR
Real-time polymerase chain reaction
Vertebrates
Host-Pathogen Interactions
Medicine
Transcription
Research Article
040301 veterinary sciences
Science
Immunology
Computational biology
Biology
Real-Time Polymerase Chain Reaction
Birds
03 medical and health sciences
Immune system
Extraction techniques
Genetics
Animals
Immune response
Poultry Diseases
Innate immune system
Organisms
Biology and Life Sciences
RNA extraction
Immunity
Innate
High-Throughput Screening Assays
Immunity
Humoral
Research and analysis methods
030104 developmental biology
Molecular biology techniques
Microfluidic
Fowl
Amniotes
RNA
Chickens
Multiplex Polymerase Chain Reaction
Spleen
Zdroj: PLoS ONE
PLOS ONE
PLoS ONE, Vol 14, Iss 12, p e0225658 (2019)
Borowska, D, Kuo, R, Bailey, R A, Watson, K, Kaiser, P, Vervelde, L & Stevens, M 2019, ' Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses ', PLoS ONE . https://doi.org/10.1371/journal.pone.0225658
ISSN: 1932-6203
Popis: To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed qPCR 96.96 Fluidigm Dynamic Array to analyse the transcription of chicken immune-related genes. This microfluidic system permits the simultaneous analysis of expression of 96 transcripts in 96 samples in 6 nanolitre reactions and the 9,216 reactions are ready for interpretation immediately. A panel of 89 genes was selected from an RNA-seq analysis of the transcriptional response of chicken macrophages, dendritic cells and heterophils to agonists of innate immunity and from published transcriptome data. Assays were confirmed to be highly specific by amplicon sequencing and melting curve analysis and the reverse transcription and preamplification steps were optimised. The array was applied to RNA of various tissues from a commercial line of broiler chickens housed at two different levels of biosecurity. Gut-associated lymphoid tissues, bursa, spleen and peripheral blood leukocytes were isolated and transcript levels for immune-related genes were defined. The results identified blood cells as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially transcribed between birds housed under varying biosecurity levels. Conventional qPCR analysis of three differentially transcribed genes confirmed the results from the multiplex qPCR array. A highly multiplexed qPCR-based platform for evaluation of chicken immune responses has been optimised and validated using samples from commercial chickens. Apart from applications in selective breeding programmes, the array could be used to analyse the complex interplay between the avian immune system and pathogens by including pathogen-specific probes, to screen vaccine responses, and as a predictive tool for immune robustness.
Databáze: OpenAIRE
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