gender: male breed: Santa Inês age: 12 months tissue: testicles treatment: none phase of infection: control
Treatment protocol
The rams were kept in an isolated pen and experimentally challenged with a rough (R) virulent B. ovis PA strain (ATCC 25840).
Growth protocol
Twelve 12-month-old healthy Santa Inês rams, previously diagnosed as negative for brucellosis/visna maedi/toxoplasmosis/neosporosis, were selected for the study.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from tissue samples collected at 0, 60, 120, 240 dpc using the Invisorb® Spin Tissue RNA Mini Kit (Invitek Gmbh, Germany) according to the manufacturer’s instructions, and purified with the RNeasy Mini-kit (Qiagen, Mississauga, ON, Canada). Total RNA was quantified by absorbance at 260 nm. The quality of all RNA preparations was controlled by electrophoresis on agarose gels. They were also evaluated on an Agilent Bioanalyser instrument (Agilent Technologies, Palo Alto, CA, USA). Only samples with a preserved rRNA ratio (28S/18S), no evidence of RNA degradation, and values ≥ 8 at the Bioanalyser were used in the microarray hybridization and qPCR.
Label
Cy3
Label protocol
RNA labeling was performed as recommended by the manufacturer.
Hybridization protocol
Expression measurements were performed using the Affymetrix GeneChip® Bovine Genome Array (Affymetrix, USA), which interrogates 23,000 genes, following the hybridization protocol recommended by the manufacturer.
Scan protocol
After array scanning, quality control was performed with GCOS software (Affymetrix, USA) according to the manufacturer’s recommendations.
Description
ctr_test.cel Gene expression data from uninfected rams.
Data processing
The quality control also was verified using Expression Console (http://www.affymetrix.com/browse/level_seven_software_products_only.jsp?productId=131414&categoryId=35623#1_1). The unwanted variables (batch effects) were removed using ComBat methodology (http://jlab.byu.edu/ComBat/Abstract.html) (Johnson et al., 2007). The IQR (Inter-Quartile-Range) filter available at R/Bioconductor (Irizarry et al., 2003) was applied. Gene expression values were obtained using the three-step Robust Multi-array Average (RMA) pre-processing method, implemented in the Affy package from R/Bioconductor (Irizarry et al., 2003). We employed the RankProd method for the selection of differentially expressed genes (DEGs), considering a p-value cut-off of 0.05 adjusted for FDR (False Discovery Rate) (Benjamini & Hochberg, 1995). RankProd is a rank-based nonparametric procedure (Hong et al., 2006). The method used HCL (Hierarchical Clustering) for the grouping of data with the Pearson correlation coefficient for the calculation of within-group distance and complete linkage between groups for the calculation. The method is available in the program Expander (http://acgt.cs.tau.ac.il/expander/index.html) (Shamir et al., 2005). We performed functional annotation analysis of the differentially expressed genes with the IPA (Ingenuity Pathway Analysis, http://www.ingenuity.com) tools. In IPA, we considered the default parameter Molecules per Network=35, Networks per Analysis=25, only direct relationships between genes and the “Ingenuity Expert Information” Data Source, including the new option of “Ingenuity ExpertAssist Findings”, in which the information has been manually reviewed and curated from full-text scientific publications.
