breed: Danish Holstein Friesian cow tissue: lobuloalveolar gland region animal number: 1586 e. coli inoculation site: Left front quarter treatment group: E.coli T=192h
Treatment protocol
Sixteen, healthy primiparous Danish Holstein-Friesian cows were challenged intra mammarily with E.coli (k2bh2) (20-40 CFU/ml) 4 to 6 weeks after parturition in one quarter. Quarters for E.coli inoculation and biopsy were selected based on CMT scores (≤ 2) and SCC in fore milk using the portable DeLaval Cell Counter (DCC; DeLaval, Tumba, Sweden) (range 1-6000 x 103 cells/ml). The front quarter with the lowest SCC (< 27,000 cells /ml) was chosen for E.coli inoculations. Control quarters where determined on the bacteriological examinations conducted prior to E.coli inoculation and on the quarter fore milk SCC at 24 h (< 181.000 cells /ml). The cows were housed in a traditional straw-bedded tiestall barn, where they were fed individually, and with free access to water. A total mixed ration (TMR) diet including vitamins and minerals was fed ad libitum twice a day in equal portions at 8.00 a.m. and 15.30 p.m. The cows were milked at 6.00 a.m. and again at 17.00 p.m. All procedures involving animals were approved by the Danish Animal Experiments Inspectorate and complied with the Danish Ministry of Justice Laws concerning animal experimentation and care of experimental animals (Buitenhuis et al. 2010: In: Proceedings of the 9th World Congress on Genetics Applied to Livestock Production, Leipzig, Germany. 1-6 August. CD-ROM communication no. 0250_PP4-167).
Extracted molecule
total RNA
Extraction protocol
Udder biopsies were collected from the infected quarter and a healthy control quarter of each cow at 24 h and 192 h after the E.coli challenge. The udder biopsies were frozen immediately in liquid nitrogen and transported to the laboratory where the tissues were stored at −80 C until RNA extraction. Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Fifteen micrograms of cRNA was fragmented at 94 C for 35 min in a final volume of 40 ul in a buffer containing 40mM Tris-acetate pH 8.1, 100mM KOAc, and 30 mM MgOAc. Next, 260 ul of 6x SSPE-T hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton) was added and the cRNA was denatured by heating to 95 C for 5 min. The hybridization mixture was loaded onto the Affymetrix probe array cartridge (Affymetrix Bovine Genome Array) and incubated for 16 hr at 45 C at constant rotation (60 rpm). The washing and staining procedure was performed in the Affymetrix Fluidics Station. The probe array was exposed to 10 washes in 6x SSPE-T at 25 C followed by 4 washes in 0.5x SSPE-T at 50 C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, final concentration 2 ug/ul (Molecular Probes, Eugene, OR) in 6x SSPE-T for 30 min at 25 C followed by 10 washes in 6x SSPE-T at 25 C. An antibody amplification step followed, using normal goat IgG as blocking reagent, final concentration 0.1 mg/ml (Sigma, St. Louis), and biotinylated anti-streptavidin antibody (goat), final concentration 3 ug/ml (Vector Laboratories, Burlingame, CA). This was followed by a staining step with a streptavidinphycoerythrin conjugate, final concentration 2 ug/ul (Molecular Probes, Eugene, OR) in 6x SSPE-T for 30 min at 25 C and10 washes in 6x SSPE-T at 25 C (Kristensen et al. 2005: Genetics 171: 157–167).
Scan protocol
The arrays were scanned at 560 nm using a confocal microscope (Hewlett Packard GeneArray Scanner G2500A)
Description
1586_VF_E.coli_192 A377-112.CEL
Data processing
The data was analyzed using R (version 2.10.0) (http://www.r-project.org/). The Bovine genome array annotation is available from the NetAffx Analysis Center (Bovine.na29.annot.csv). Additional and updated annotation was obtained from the Ensemble database, using the biomaRt package (version 2.0.0) in R. Normalization of the expression values for the udder samples were performed using the Robust Multi-array Average (RMA) algorithm as implemented in the Affy package (version 1.24.2).Differential expression of each gene was assessed using linear modeling and empirical Bayes methods, which were implemented using the R package Limma (version 3.2.1). The linear models allowed for changes for time points. The contrasts tested were 24 h p.i. vs. 24 h control (T24 – C24) and 192 h p.i. vs. 192 h control (T192 – C192), respectively. Each transcript targeted by a probe was tested for its expression change using a modified t-test. In the modified t-test, the residual standard deviations are moderated across the probe sets to ensure that there is a more stable inference for each transcript. The moderated standard deviations are a compromise between the individual transcript-wise standard deviations and the pooled standard deviation. Multiple testing was accounted for using the bonferroni correction. Probes were considered differentially expressed (DE) if the corrected P-value was below 0.05 and had a minimum log2-fold change of 1 for both T=24h and T=192h. A hyper geometric gene-set enrichment test (GOstats package version 2.12.0) was performed based on the clusters identified in at T=24h and T=192h, separately. Overrepresentation of gene sets defined by the group of biological processes (BP) in the Gene Ontology database (GO) (http://www.geneontology.org/) or the Kyoto Encyclopedia of Genes and Genomes database (KEGG) (http://www.genome.jp/kegg/) was tested using the Fisher’s exact test. For this test only the significant genes which were annotated with an Entrez gene id were included. When a gene had a duplicate on the array only one gene id was used. A gene-set was considered significant if P < 0.05.
