|
| Status |
Public on Jan 02, 2016 |
| Title |
Lung_HPAIV (H5N1) Clade 2.2_Bird2 |
| Sample type |
RNA |
| |
|
| Source name |
Lung_5dpi_HPAIV (H5N1) Clade 2.2
|
| Organism |
Anas platyrhynchos |
| Characteristics |
age: 6 weeks
challenged with: HPAIV (H5N1) Clade 2.2
time point: 5 dpi
tissue: lung
|
| Treatment protocol |
Six weeks old AIV seronegative domestic ducks were divided into three groups and each group containing 4 birds. In group 1ducks was intranasally inoculated with 106 EID50 of HPAI H5N1 virus isolate and the group 2 was inoculated with 106 EID50 of HPAI H5N1 virus isolate. The group 3 was inoculated with PBS. Birds were observed daily for clinical signs. The birds were sacrificed from all groups at 5 days of post infection (dpi) and lung tissues collected in RNA latter and stored at -800C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolated from the lung tissues of three birds from each infected group and two birds from non-infected group. Total RNA was isolate using TRIzol® Reagent (Invitrogen, USA) with the Qiagen’s RNeasy mini kit (Qiagen, Germany). The integrity of RNA was analyzed on the Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
The sample labeling was performed using Quick-Amp Labeling Kit, One Color (Agilent Technologies, Part Number: 5190-0442). 500ng of total RNA was denatured along with appropriate diluted Spike In controls; Spike A Mix/cyanine 3-CTP (Agilent Technologies, RNA Spike In Kit, One Color, Part Number 5188-5282) and oligodT primers tagged to T7 promoter region at 65°C for 10 min followed by snap chilling on ice for 5 minutes.The cDNA master mix was added to the denatured RNA sample and incubated at 40°C for 2 hours.The cDNA sample mix was denatured at 65°C for 15 min to denature the enzymes in the cDNA reaction. The in vitro transcription master mix was added to the cDNA samples and incubated at 40°C for 2.30 hours. Cy3-CTP present in the reaction mix gets incorporated into the newly synthesized strands. The Cyanine 3-CTP labeled cRNA samples were purified using Qiagen RNeasy column (Qiagen, Cat No: 74106). The concentration of cRNA and dye incorporation was determined using Nanodrop-1000 (JH Bio) and the specific activity was estimated.
|
| |
|
| Hybridization protocol |
600ng of the labeled Cyanine 3-CTP cRNAs were fragmented to an average size of approximately 50 to 100 nucleotides with fragmentation buffer at 60°C for 30 minutes and the reaction was stopped by adding 2X GE HI-RPM hybridization buffer (Agilent Technologies, In situ Hybridization kit, Part Number5190-0404).The samples were pipetted onto the gasket slide and hybridized on to arrays .The hybridization was carried out at 65°C for 16 hours.After hybridization, the slides were washed using Gene Expression Wash Buffer1 (Agilent Technologies, Part Number 5188-5325) at room temperature for 1 minute and Gene Expression Wash Buffer 2 (Agilent Technologies, Part Number 5188-5326) at 37 oC for 1 minute.
|
| Scan protocol |
The microarray slide was scanned using Agilent Scanner (Agilent Technologies, Part Number G2600D).
|
| Description |
Gene expression after 5dpi in HPAIV (H5N1) clade 2.2 virus-challenged duck lung tissue
BB2
|
| Data processing |
Data extraction from Images was done using Feature Extraction software and Normalization of the data was done in GeneSpring GX : 75th percentile shift method
|
| |
|
| Submission date |
Jan 23, 2015 |
| Last update date |
Jan 02, 2016 |
| Contact name |
Anamika Mishra |
| E-mail(s) |
reach2anamika@yahoo.com
|
| Phone |
08015703570
|
| Organization name |
NIHSAD
|
| Department |
Animal Genetics
|
| Lab |
Pathogenomics lab
|
| Street address |
Anand Nagar
|
| City |
bhopal |
| State/province |
Madhya Pradesh |
| ZIP/Postal code |
462022 |
| Country |
India |
| |
|
| Platform ID |
GPL19666 |
| Series (1) |
| GSE65230 |
Genome wide host gene expression analysis in duck lungs infected with highly or low pathogenic H5N1 avian influenza virus |
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