Unknown,Transcriptomics,Genomics,Proteomics

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Comparative analysis of chicken Intestinal Intraepithelial lymphocytes (IEL) following Eimeria acervulina (EA), E. maxima (EM), or E. tenella (ET) infection

ABSTRACT: Relative expression levels of mRNAs in chicken IEL experimentally infected with EA, EM, or ET were measured at 1 to 6 days post-infection (dpi) following primary and secondary infections. One week-old chickens were uninfected (negative control) or were orally inoculated with sporulated oocysts of EA, EM, or ET. One week later, the infected chickens were challenged with an identical inoculum of the homologous parasite. Intestinal samples were collected daily from 5 birds in a treatment group at from 1 to 6 dpi following primary and secondary infections. Cecum, duodenum, and jejunum were collected from the birds challenged with E. acervulina, E. maxima, and E. tenella, respectively. Uninfected control samples and one of the 3 infection group samples were labeled with different fluorescent dyes and hybridized simultaneously on the same slide using a reference design with a dye swap protocol. Thirty seven-condition experiment, Non-infected control vs. Primary or secondary EA, EM, or ET infected IEL at 1 to 6. Biological replicates: 2 replicates with dye-switching from each infection groups. Two replicates per array.

ORGANISM(S): Gallus gallus

SUBMITTER: Duk Kyung Kim

PROVIDER: E-GEOD-31213 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:One day-old broilers (Ross/Ross, Longenecker's Hatchery, Elizabethtown, PA) were housed in Petersime starter brooder units and randomly assigned to 4 groups (5 birds/group). Carvacrol (Nature Chemicals, Hamburg, Germany) and cinnamaldehyde (Alys Technologies SA, Bussigny-près-Lausanne, Switzerland) were obtained from the compound obtained from synthesis. Crushed C. annuum fruits (Pushp Brand Spices, Munimji Foods and Spices Pvt. Ltd. Indore, India) were extracted with volatile solvents using the percolation method to obtain oleoresin. Chickens were fed for 7 days beginning from hatch with a standard diet alone (control) or with diets supplemented with carvacrol, cinnamaldehyde, or Capsicum oleoresin.<br> Following euthanization of the birds, intestines were cut longitudinally and washed three times with ice-cold Hanks? balanced salt solution (HBSS) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma, St. Louis, MO). The mucosal layer of entire intestine was carefully scraped using a surgical scalpel and intraepithelial lymphocytes (IELs) were isolated by Percoll density gradient centrifugation as previously described (Min et al. 2005). Total RNA was isolated from 5.0 x 107 cells using Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA). Aminoallyl-labeled RNA from IELs was prepared using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion, Austin, TX). Two of 20 ?g aliquots of each aminoallyl-RNA sample were fluorescently labeled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen) and labeled RNAs were column-purified using the RNA Amplification Kit (Ambion). RNA concentrations and labeling efficiencies were determined spectrophotometrically.<br> Hybridizations were performed using HybIt hybridization buffer (TeleChem, Sunnyvale, CA) in ArrayIt reaction cassettes at 50°C overnight as described (Kim et al., 2008). After hybridization, the slides were rinsed in 0.5 X SSC, 0.01% SDS at room temperature and washed once for 15 min in 0.2 X SSC, 0.2% SDS at 50°C, 3 times for 1 min in 0.2 X SSC at room temperature, and 3 times for 1 min in distilled water at room temperature. Each sample had a repeated hybridization using the alternate fluorescent dye between the treatment and control.

Dataset Information

Comparative analysis of chicken Intestinal Intraepithelial lymphocytes (IEL) following Eimeria acervulina (EA), E. maxima (EM), or E. tenella (ET) infection

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