|
| Status |
Public on Mar 01, 2012 |
| Title |
Non-vaccinated, challenged, day 5, mild, replicate 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chicken PBL non-vaccinated, non-challenged, day 1
|
| Organism |
Gallus gallus |
| Characteristics |
Sex: male
cell type: peripheral blood leukocytes
vaccine status: Non-vaccinated
challenge status: Non-challenged
time: Day 1
pathology: n/a
infection: uninfected
|
| Treatment protocol |
Male broilers chicks were vaccinated/non-vaccinated at 2 weeks of age and challenged/non-challenged at 4 weeks of age. Necropsies took place 1 and 5 days post-challenge, at which time blood was taken and lesion scores assessed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using MagMax-96 for microarrays
|
| Label |
Cy3
|
| Label protocol |
400ng of total RNA was primed by adding T7 Promoter primer and incubating at 65C for 10 minutes. RNA was reverse-transcribed into cDNA by using RT enzyme and dNTP mix, incubating at 40C for 2 hours followed by 65C for 15 minutes. cDNA was transcribed into cRNA which was labeled with either Cy3 or Cy5 dye, then purified using RNeasy kit.
|
| |
|
| Channel 2 |
| Source name |
Chicken PBL non-vaccinated, challenged, day 5, mild
|
| Organism |
Gallus gallus |
| Characteristics |
Sex: male
cell type: peripheral blood leukocytes
vaccine status: Non-vaccinated
challenge status: Challenged
time: Day 5
pathology: Mild
infection: Avian Pathogenic Escherichia coli
|
| Treatment protocol |
Male broilers chicks were vaccinated/non-vaccinated at 2 weeks of age and challenged/non-challenged at 4 weeks of age. Necropsies took place 1 and 5 days post-challenge, at which time blood was taken and lesion scores assessed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using MagMax-96 for microarrays
|
| Label |
Cy5
|
| Label protocol |
400ng of total RNA was primed by adding T7 Promoter primer and incubating at 65C for 10 minutes. RNA was reverse-transcribed into cDNA by using RT enzyme and dNTP mix, incubating at 40C for 2 hours followed by 65C for 15 minutes. cDNA was transcribed into cRNA which was labeled with either Cy3 or Cy5 dye, then purified using RNeasy kit.
|
| |
|
| |
| Hybridization protocol |
Blocking agent, fragment buffer and hybridization buffer were added to 825ng of Cy3 and 825ng Cy5 labeled cRNA. Samples were applied to a gasket slide and hybridized to the microarray slide. After hybridization, slides were washed in wash buffer then a stabilization agent
|
| Scan protocol |
Slides scanned using GenePix 4100A scanner
Images quantified using GenePix Pro v6.0
|
| Data processing |
Probes with an average Signal to Noise ratio of less than 3 over all slides were excluded from further study, where SNR is (Median Foreground - Median Background) / Standard Deviation of Background. Median background was subtracted from median foreground, LOWESS normalization was done using R program
|
| |
|
| Submission date |
Aug 15, 2011 |
| Last update date |
Mar 01, 2012 |
| Contact name |
Susan J Lamont |
| E-mail(s) |
sjlamont@iastate.edu
|
| Organization name |
Iowa State University
|
| Department |
Animal Science
|
| Street address |
2255 Kildee Hall
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL6413 |
| Series (1) |
| GSE31387 |
Transcriptome Response of Leukocytes from Chickens Infected with Avian Pathogenic Escherichia coli Identifies Pathways Associated with Resistance |
|
