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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.
This article was published in the following journal.
Name: PloS one
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A quantitative prediction of the biological, ecotoxicological or pharmaceutical activity of a molecule. It is based upon structure and activity information gathered from a series of similar compounds.
Locations, on the GENOME, of GENES or other genetic elements that encode or control the expression of a quantitative trait (QUANTITATIVE TRAIT, HERITABLE).
A ubiquitously-expressed cysteine peptidase that exhibits carboxypeptidase activity. It is highly expressed in a variety of immune cell types and may play a role in inflammatory processes and immune responses.
The type species of GYROVIRUS, a small, non-enveloped DNA virus originally isolated from contaminated vaccines in Japan. It causes chicken infectious anemia and may possibly play a key role in hemorrhagic anemia syndrome, anemia dermatitis, and blue wing disease.
A characteristic showing quantitative inheritance such as SKIN PIGMENTATION in humans. (From A Dictionary of Genetics, 4th ed)
Polymerase Chain Reaction (PCR)
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Immunoassay - ELISA
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A diagnostic test is any kind of medical test performed to aid in the diagnosis or detection of disease. For example: to diagnose diseases to measure the progress or recovery from disease to confirm that a person is free from disease Clin...