|
| Status |
Public on Sep 01, 2008 |
| Title |
E1A2- AC to AI |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Salmonella enteritidis resistant line AC not infected
|
| Organism |
Gallus gallus |
| Characteristics |
Heterophils from 1 wk chicken
|
| Treatment protocol |
Heterophils (1×10^7) were treated with 300 µl RPMI for 30 min at 39°C on a rotary shaker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Treated heterophils were pelleted, washed with RPMI (485×g for 15 min at 4°C), the supernatant discarded, the cells re-suspended in lysis buffer (Qiagen RNeasy mini RNA extraction kit, Qiagen Inc., Valencia, CA), and frozen. The lysed cells were transferred to QIAshredder homogenizer columns and centrifuged for 2 min at _ 8000 × g. Total RNA was extracted from the homogenized lysate according to the manufacturer’s instructions, eluted with 50 µl RNase-free water and stored at -80°C.
|
| Label |
Cy5
|
| Label protocol |
A 500 ng of aliquot of total RNA was reverse transcribed into cDNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). The synthesized cDNA was transcribed into cRNA labeled with one of two cyanine-labeled nucleotides (Perkin Elmer, Wellesley, MA). Labeled cRNA was purified with RNeasy Mini columns (Qiagen, Valecia, CA). The quality of each cRNA sample was verified by total yield and specificity measured with ND-100 spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
Salmonella enteritidis resistant line AI treated with infection
|
| Organism |
Gallus gallus |
| Characteristics |
Heterophils from 1 wk chicken
|
| Treatment protocol |
Heterophils (1×10^7) were treated with 300 µl SE (#97-11771) for 30 min at 39°C on a rotary shaker.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Treated heterophils were pelleted, washed with RPMI (485×g for 15 min at 4°C), the supernatant discarded, the cells re-suspended in lysis buffer (Qiagen RNeasy mini RNA extraction kit, Qiagen Inc., Valencia, CA), and frozen. The lysed cells were transferred to QIAshredder homogenizer columns and centrifuged for 2 min at _ 8000 × g. Total RNA was extracted from the homogenized lysate according to the manufacturer’s instructions, eluted with 50 µl RNase-free water and stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
A 500 ng of aliquot of total RNA was reverse transcribed into cDNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). The synthesized cDNA was transcribed into cRNA labeled with one of two cyanine-labeled nucleotides (Perkin Elmer, Wellesley, MA). Labeled cRNA was purified with RNeasy Mini columns (Qiagen, Valecia, CA). The quality of each cRNA sample was verified by total yield and specificity measured with ND-100 spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
A 825 ng of labeled cRNA with specificity over 8 were used for the hybridization reaction using the in situ hybridization kit plus (Agilent Technologies). A 100 ul hybridization solution was loaded on each of array in Agilent 4x44K microarrays. Arrays were incubated at 65 ℃ for 17h in Agilent’s microarray hybridization chambers. After hybridization, arrays were washed according to the Agilent protocol.
|
| Scan protocol |
Slides were scanned at 5-μm resolution using GenePix Personal 4100A (Molecular Devices Corporation, Sunnyvale, CA) and were saved as TIFF images. Auto Photomultiplier tube (PMT) were selected and adjusted to get the ratio of the overall intensities between the two channels (Cy3 and Cy5) was 0.9~1.1. The intensities of the spots on each image were quantified by Genepix pro 6.0 software (Molecular Devices Corporation, Downingtown, PA), and data were saved as .txt files for further analyses.
|
| Description |
Four arrays are included in one slide
|
| Data processing |
The signal intensity of each gene was globally normalized using LOWESS by R program (Yang et al, 2002).
|
| |
|
| Submission date |
Oct 23, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Huaijun Zhou |
| Organization name |
Texas A&M University
|
| Street address |
Rm 418D Kleberg Center,Texas A&M University
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-2472 |
| Country |
USA |
| |
|
| Platform ID |
GPL4993 |
| Series (1) |
| GSE9416 |
Gene expression profiling in heterophils with Salmonella enteritidis challenge using a chicken 44K Agilent microarray |
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