Abstract
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.
Highlights
Poultry are vital to global food security and the Food and Agriculture Organisation of the United Nations estimated that 75 billion broilers and 1.9 trillion eggs were produced in 2016
To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed quantitative PCR (qPCR) 96.96 Fluidigm Dynamic Array to analyse the transcription of chicken immune-related genes
This type of high-throughput platform would be advantageous for the rapid and cost-effective analysis of immune responses, as a diagnostic tool to screen for pathogens and as a selection tool to breed more robust chicken lines based on production traits supported by desired gene expression profiles
Summary
Poultry are vital to global food security and the Food and Agriculture Organisation of the United Nations estimated that 75 billion broilers and 1.9 trillion eggs were produced in 2016. Screening hundreds of targets and samples in parallel quantitative PCR (qPCR) for gene expression is possible with high-throughput qPCR tools, for example the 96.96 Dynamic Array from Fluidigm [2] or NanoString nCounter gene expression system [3]. This type of high-throughput platform would be advantageous for the rapid and cost-effective analysis of immune responses, as a diagnostic tool to screen for pathogens and as a selection tool to breed more robust chicken lines based on production traits supported by desired gene expression profiles
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