tissue: Thalamus exposure to wnv: No vaccination for wnv: N/A
Treatment protocol
Horses in the vaccinated and exposed group were given IM injections of two doses of modified live attenuated Yellow Fever (YF) chimera vaccine for protection against WNV (Prevenile, Intervet-Schering-Plough) 28 days apart, and challenged with WNV/NY/99 crow at a dose of 1 x 10^5 PFU/mL intrathecally 28 days after the last vaccination. Horses in the non-vaccinated and exposed group were given diluent 28 days apart and challenged with WNV/NY/99 crow at a dose of 1 x 10^5 PFU/mL intrathecally 28 days after the last diluent dose. Horses in both groups were monitored daily for changes in temperature and clinical signs including neurological symptoms (ataxia, paresis, changes in mentation, etc). Horses in these groups were euthanized (University of Florida IACUC protocols #F077, #F093, #D163) if demonstrating clinical signs for humane reasons or at the end of the study (day 21) if not demonstrating clinical signs. Normal control horses were not exposed to WNV and were euthanized for humane reasons after surrender by owner (lameness and age). These horses underwent thorough physical and neurological exams and were not demonstrating any signs of neurological or immunological disease at the time of euthanasia.
Extracted molecule
total RNA
Extraction protocol
All horses were necropsied immediately upon euthanasia. Tissues were snap frozen in dry ice and ethanol and stored at -80oC until RNA extraction was performed. RNA was extracted using Trizol (Invitrogen) and a phase separation technique according to manufacturers' protocol. The only modification was an added chloroform extraction technique to remove extra lipids from brain tissue. RNA quality was assessed using spectrophotometric technology (ND-1000, Nanodrop Technologies, Wilmington, DE) and a microfluidics platform (Agilent 2100 Bio-analyzer, Santa Clara, CA). Only samples with a 260:280 ratio> 1.8 and a RIN number >6 were used.
Label
Cy3
Label protocol
Dye-labeled cDNA was created using Cy3 dye (One-Color Microarray-Based Gene Expression Analysis kit, Agilent Technologies) according to the manufacturer’s protocols.
Hybridization protocol
Hybridization to the microarrays was performed according to the manufacturer’s protocol. Individual, non-pooled cDNA samples were hybridized to the arrays. Briefly, the prioprietary blocking agent was prepared to a 10X concentration and incubated at 37C for 5 minutes. Individual tubes were prepared combining 1.65 mg of dye-labeled cDNA, 11 mL of 10X blocking agent, nuclease-free water, and 2.2 mL of the proprietary 25X fragmentation buffer for a final volume of 55 mL. The tubes were incubated at 60C for 30 minutes and then placed on ice for 1 minute. Fifty mL of the proprietary 2X hybridization buffer was added to each tube. The samples were mixed, centrifuged at 13,000 rpm for 1 minute at 25C, and placed on ice. The gaskets were placed into the chambers and 100 mL of sample added to each chamber. The array slides were placed on top of each gasket. The chambers were closed in the hybridization oven and rotated at 10 rpm for 17 hours at 65oC. After the hybridization, the arrays were disassembled in wash buffer 1. The slides were washed for 1 minute in proprietary wash buffers at 37oC. The excess liquid was dried off and the slides washed for 30 seconds in the proprietary stabilization and drying solution.
Scan protocol
Slides were scanned on the Agilent DNA Microarray Scanner using one color scan setting (dye channel set to green) for 1x44k array slides (65 micron feature size).
Description
Gene expression in normal, healthy controls not exposed to West Nile virus
Data processing
Scanned images were analyzed with proprietary software (Feature Extraction Software, Agilent) using default parameters. Features that were tagged as Feature Non-uniform outliers were excluded. Agilent spike-in controls were used and background subtraction performed.
