|
| Status |
Public on Dec 09, 2020 |
| Title |
252644011436: IFN Treated pig # 9_dpi 6_PBMC |
| Sample type |
RNA |
| |
|
| Source name |
PBMC samples from 6 days after innoculation of Ebola Kikwit from pig 9
|
| Organism |
Sus scrofa |
| Characteristics |
sample_id: 17
tissue: PBMC
gender: Male
age: 6 weeks
|
| Treatment protocol |
7 pigs were randomly divided in 2 groups: A (4 pigs) and B (3 pigs). Animals in Group A (# 7, 8, 9, 10) were inoculated subcutaneously (SC) in the neck with 2x1010 pfu/2ml of Ad5-porIFNα. Animals in Group B (Control group #11, 17, 18) were SC inoculated with 2 ml of phosphate-buffered saline (PBS). One day later all pigs were oro-nasally inoculated with 106 pfu/2ml/pig of EBOV (0.5 ml per each nostril, 1ml orally). Arbitrarily, the day of inoculation (dpi) with EBOV was designated as 0 dpi. Clinical signs, behavioral changes and rectal temperature were recorded daily. Nasal washes, oral swabs and blood were collected for each pig the day of Ad5-porIFNα or PBS treatment (-1 dpi), and at 0, 3 and 5 or 6 dpi with EBOV. Blood was collected into BD vacutainer CPTTM cell preparation tubes (Becton Dickinson, New Jersey, USA), plasma and the peripheral blood mononuclear cells (PBMC) were separated according to the manufacturer’s protocol. PBMC pellets diluted four times with PBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PBMC and BALP pellets and the concentrations were determined using a NanoDrop (NanoDrop Technologies) and shipped to MOGENE LC, St. Louis, MO, USA for the microarray assay.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification. Dye incorporation and cRNA yield were checked with the NanoDrop.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Porcine V2 arrays (G2519F-026440) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Microarray slides were scanned on Agilent high resolution C scanner (G2505C) and images processed using the Agilent Feature Extraction software. Quality of samples was analyzed using the ArrayQualityMetrics package and the data imported into GeneSpring 14.8 (Agilent) for further analysis.
|
| Description |
Gene expression in PBMC samples 6 days after innoculation of Ebola Kikwit from pig pre-treated with IFN
|
| Data processing |
The data was normalized using a shift to the 75th percentile; thereafter the value was further transformed as log2
|
| |
|
| Submission date |
Dec 08, 2020 |
| Last update date |
Dec 09, 2020 |
| Contact name |
Chandrika Senthilkumaran |
| E-mail(s) |
[email protected]
|
| Phone |
4162778590
|
| Organization name |
Canadian food inspection agency
|
| Lab |
Chandrika Senthilkumaran
|
| Street address |
8, Wisteria RD
|
| City |
toronto |
| State/province |
ON |
| ZIP/Postal code |
M1R 4X8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16571 |
| Series (1) |
| GSE162846 |
Effect of Ad5-porcine interferon-α pre-treatment in Ebola virus induced gene expression in pigs |
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