Each necropsy specimen was macerated in a laboratory blender and 0.5 - 0.8 ml of tissue suspension was seeded onto each of two culture plates of the modified Thayer-Martin's medium. The plates were read after 4 and 7 days of incubation at 37 ºC in 10% CO2. The Brucella colonies were identified by colonial morphology, Gram staining, oxidase and urease tests, CO2 requirement and phage typing.
Growth protocol
The rams were challenged with a total dose of 1.7 x 109 B. ovis CFU, as shown by viable cell counts made on the day of inoculation, in 60 l doses (30 l administered conjunctivally and 30 l intrapreputially).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from Buffy coat samples of experimentally infected and control sheep using TriReagent (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions. The RNA was checked using the Experion™ Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA) to evaluate the quality and integrity of RNA preparations.
Label
Cy5 Fluor
Label protocol
One µg total RNA was labeled using the 3DNA Array900MPX kit with Cy5 and Cy3 fluor dyes (Genisphere, Hatfield, PA, USA), the supplied random and oligo-dT primes, Superscript II (Invitrogen, Carlsbad, CA, USA), the supplied formamide-based hybridization buffer and 24x60 mm LifterSlips slides (Erie Scientific, Portmouth, NH, USA) according to the manufacturer’s (Genisphere) instructions.
Lack of B. ovis infection was assessed by culture.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from Buffy coat samples of experimentally infected and control sheep using TriReagent (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions. The RNA was checked using the Experion™ Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA) to evaluate the quality and integrity of RNA preparations.
Label
Cy3 Fluor
Label protocol
One µg total RNA was labeled using the 3DNA Array900MPX kit with Alexa Fluor dyes (Genisphere, Hatfield, PA, USA), the supplied random and oligo-dT primes, Superscript II (Invitrogen, Carlsbad, CA, USA), the supplied formamide-based hybridization buffer and 24x60 mm LifterSlips slides (Erie Scientific, Portmouth, NH, USA) according to the manufacturer’s (Genisphere) instructions.
Hybridization protocol
Labeled total RNA (1 µg) was hybridized to the microarrays using the supplied formamide-based hybridization buffer and 24x60 mm LifterSlips slides (Erie Scientific, Portmouth, NH, USA) according to the manufacturer’s (Genisphere) instructions. Post-hybridization washes were done according to the manufacturer’s (Genisphere) instructions.
Scan protocol
Hybridization signals were measured using a ScanArray Express (PerkinElmer, Boston, MA, USA) and the images where processed using GenePix Pro version 4.0 (Axon, Union City, CA, USA).
Description
Normalized (global median) ratio values obtained for each probe were averaged across replicates. Significant differences were defined as those displaying an average expression fold change greater than 2-fold (log (base 2) ratio ≥ 1.0).
Data processing
Normalization of log2 ratios using global median normalization
Submission date
Jun 28, 2008
Last update date
Jun 30, 2008
Contact name
Ruth Cecilia Galindo
Organization name
Instituto de Investigacion en Recursos Cinegéticos
