|
| Status |
Public on Jul 01, 2016 |
| Title |
PBMC_2h_TC |
| Sample type |
RNA |
| |
|
| Source name |
PBMC, Indigenous Control, 2h
|
| Organism |
Bos indicus |
| Characteristics |
tissue: PBMC
gender: Male
stress: none
|
| Treatment protocol |
Peripheral blood mononuclear cells (PBMC) were separated from aseptically collected blood under cold conditions by density gradient centrifugation (Histopaque-1.083, Sigma). Thereafter, one half of each PBMC sample was infected with T. annulata (Parbhani strain) sporozoite preparations. In brief, the cells were resuspended at 2x106 cells/ml in RPMI-1640 medium supplemented with 20% FBS and aliquoted into a 6-well plate. An equal volume of sporozoite suspension at 0.5 tick equivalents/ml in RPMI-1640 medium supplemented with 40% FBS was added to the PBMC and incubated at 37 C in a 5% CO2 incubator for 2 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 2 h of incubation, PBMC culture was harvested from plates in 15ml centrifuge tubes and supernatant was removed after centrifugation at 1500 rpm for 10 min. RNA isolation was done by using RNeasy Plus Mini Kit (Qiagen) as per the manufacturer’s instructions with slight modifications. The RNA was quantified by NanoDrop ND 1000 Spectrophotometer (Thermo Scientific, USA) and RNA integrity was determined by 2100 Bioanalyzer (Agilent Technologies, USA).
|
| Label |
Cy3
|
| Label protocol |
200 ng of total RNA was used to prepare Cyanine-3 (Cy3) labeled cRNA for hybridization. One-Color Low input Quick Amp labelling Kit (Agilent) was used for labeling followed by cleaning using RNeasy column purification kit (Qiagen). Dye incorporation and cRNA yield were determined with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
. For chip hybridization, 1.65 μg of Cy3 labelled cRNAs was fragmented at 60 °C for 30 min in a reaction volume of 55 μl containing 25× Agilent Fragmentation buffer and 10× Agilent Gene Expression Blocking agent. On completion, 55 μl of 2×HI-RPM hybridization buffer (Agilent) was added. Out of total volume, 100 μl of samples were hybridized on Bovine (V2) Gene Expression Microarray 4x44K, for 17 h at 65 °C in a rotating hybridization chamber (Agilent).
|
| Scan protocol |
Following hybridization, microarray slides were washed with Gene Expression wash buffer 1 (Agilent) for 1 min at room temperature and with Gene Expression wash buffer 2 (prewarmed at 37 °C for overnight) at 37 °C for 1 min. Microarray slides were scanned with an Agilent SureScan Microarray Scanner.
|
| Description |
Gene expression after 2hr in PBMC of Indigenous cattle
|
| Data processing |
The scanned images were analysed with Agilent Feature Extraction Software 11.0.1.
|
| |
|
| Submission date |
May 13, 2016 |
| Last update date |
Jul 01, 2016 |
| Contact name |
Amod Kumar |
| E-mail(s) |
amodvet@gmail.com
|
| Organization name |
Indian Veterinary Research Institute
|
| Street address |
Izatnagar
|
| City |
Bareilly |
| ZIP/Postal code |
243122 |
| Country |
India |
| |
|
| Platform ID |
GPL11648 |
| Series (1) |
| GSE81419 |
Genome-wide transcriptional profiling of peripheral blood mononuclear cells challenged with Theileria annulata in crossbred and indigenous cattle of India |
|
