Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomes of whole blood and PBMC in chickens.

ABSTRACT: The gene expression profiles of chicken whole blood (WB) and peripheral blood mononuclear cells (PBMC) have been characterized and compared by microarray analysis. This study highlights the respective advantages of each blood cell subset and show that whole blood is more suitable for large scale studies and allows for the exploration of biological processes which are so far poorly investigated. Whole blood and PBMC from four male broilers (9 weeks old) were collected and isolated before -80Â°C conservation.

ORGANISM(S): Gallus gallus

SUBMITTER: Colette DESERT

PROVIDER: E-GEOD-65331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptomes of whole blood and PBMC in chickens.

Désert Colette C Merlot Elodie E Zerjal Tatiana T Bed'hom Bertrand B Härtle Sonja S Le Cam Aurélie A Roux Pierre-François PF Baeza Elisabeth E Gondret Florence F Duclos Michel Jacques MJ Lagarrigue Sandrine S

Comparative biochemistry and physiology. Part D, Genomics & proteomics 20160627

Global transcriptome analysis of chicken whole blood to discover biomarkers of different phenotypes or physiological disorders has never been investigated so far. Whole blood provides significant advantages, allowing large scale and non-invasive sampling. However, generation of gene expression data from the blood of non-mammalian species remains a challenge, notably due to the nucleated red blood cells, hindering the use of well-established protocols. The aim of this study was to analyze the rel ...[more]

PMID: 27442111

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Project description:Peripheral blood mononuclear cells (PBMC) from cows with experimentally induced hypocalcaemia or spontaneous milk fever were subject of an oligo-microarray analysis following quantitative real-time reverse transcription PCR (q-PCR). In experimental hypocalcaemia induced by 10 % Na2EDTA infusion (n = 4), the microarray revealed that 32 genes were significantly up- or down-regulated compared to control treatment by 11 % CaEDTA infusion (n = 4). In milk fever cases (n = 8), the microarray demonstrated that 98 genes were expressed differentially higher or lower compared to healthy parturient cows (n = 5). From both data, five genes were selected to be strongly related to both experimental hypocalcaemia and milk fever. Additionally, another gene was judged to be specific for milk fever independent of hypocalcaemia. The mRNA expressions of these six genes in milk fever cases were verified by q-PCR, which were significantly higher or lower than those in healthy parturient cows. In the experimental hypocalcemia study, four healthy nonpregnant, non-lactating ovariectomised Holstein cows were used. The experiment was performed according to a 2 M-CM-^W 2 crossover design with 2 weeks intervals. Cows were infused with either Na2EDTA (hypocalcaemic treatment) or CaEDTA (control treatment) solution during a period of 4 h. The animals were monitored for clinical signs of hypocalcaemia (dry nose, staggering, astasia, reduced ruminal activity, increased heart rate, increased respiratory frequency, sweating, and ear cold). Infusion of EDTA solution was temporarily suspended when hypocalcaemia induced astasia was represent. If affected cow stood again, EDTA infusion was resumed. PBMC samples were collected before the start of infusion (0 h; pre-treatment) and at 4 and 24 h after the start of infusion 24h and utilized for gene expression analysis of PBMC. In the spontaneous milk fever study, a total of 13 Holstein dairy cows, which were 8 parturient cows diagnosed with milk fever within 2 days postpartum and 5 clinically healthy parturient cows within 2 days after parturition (Group H), were used. All spontaneous milk fever cases showed hypocalcaemia (< 1.8 mmol/l) in the blood samples obtained before the first treatment, and were treated by an intravenous infusion of a 20 % Ca borogluconate solution (500 ml). The milk fever cases were divided into two categories according to the reponse of the cows to the first Ca treatment: an immediate response in which the cow could stand within 1 day after a single Ca treatment (Group A, n = 4) and a requirement further treatment over 1 day after a single Ca treatment (Group B, n = 4). PBMC samples were collected vein just before the first Ca treatment in groups A and B and within 2 days postpartum in group H and utilized for gene expression analysis of PBMC.

Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.

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Transcriptomes of whole blood and PBMC in chickens.

Publications

Transcriptomes of whole blood and PBMC in chickens.

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