|
| Status |
Public on Aug 09, 2006 |
| Title |
Chicken day six liver, Vehicle Control rep6 vs. reference RNA pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chicken liver, day six of treatment
|
| Organism |
Gallus gallus |
| Characteristics |
Chicken liver, day six of treatment
|
| Biomaterial provider |
Moyer's Hatchery (Quakertown, PA)
|
| Treatment protocol |
At 29 days of age, broiler cockerels (Ross x Cobb) were given a subcutaneous implant of an osmostic minipump (Alzet, model 2001; ALZA, Mountain View, CA) to continuously release hormone for seven days at a rate of 1.0 microliter/hour. Birds received a vehicle (50% DMSO and 50% propylene glycol) as control (VC), exogenous corticosterone (600 micrograms CS/kg/day), exogenous L-tri-idothyronine (192 micrograms T3/kg/day), or both CS and T3. After six days of treatment, liver tissue was excised immediately, snap frozen in liquid nitrogen, and stored in -80 degrees C until RNA isolation.
|
| Growth protocol |
Chicks were fed a broiler starter ration and raised in a controlled-environment animal room under a 20-h light:4-h darkness photoperiod in a heated battery-brooder until 3 weeks of age. At 3 weeks of age, 72 birds of uniform body weight (within 1 SD) were randomly assigned to wire cages within two identical controlled-temperature rooms with three birds per pen. Rooms were maintained under the same 20L:4D light/dark cycle throughout the duration of the experiment. The birds were provided with water and a commercial grower/finisher ration ad libitum until the end of the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from liver samples of birds that received six days of treatment was isolated using a RNeasy Midi kit (Qiagen, Valencia, CA) following the manufacturer's protocol with some modifications. The quantity of extracted total RNA was determined using a NanoDrop spectrophotometer (Wilmington, DE), and the quality of extracted total RNA was examined by microcapillary electrophoresis on a BioAnalyzer 2100 (Agilent, Wilmington, DE), where the rRNA ratio (28S/18S) was observed for integrity.
|
| Label |
Alex Fluor 555
|
| Label protocol |
Chicken cDNA targets were synthesized with the SuperScript? Plus Indirect cDNA Labeling System (Invitrogen). First, total RNA samples (20 micrograms) were reverse transcribed using an anchored oligo(dT)20 primer, amino-modified dNTPs, and SuperScript III Reverse Transcriptase in 30 microliter volume. Next, the RNA template was destroyed by incubation with sodium hydroxide at 70 degrees C. First strand cDNA was purified with a Low-Elution Volume Spin Cartridge and then labeled with Alexa Fluor 555 (green) in a fluorsecent dye coupling reaction. Dye-incorporated cDNAs were purified to remove un-reacted dye and analyzed for their labeling efficiency with a NanoDrop spectrophotometer.
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| |
|
| Channel 2 |
| Source name |
Reference pool of hepatic RNA from five-week-old control chickens
|
| Organism |
Gallus gallus |
| Characteristics |
Reference pool of hepatic RNA from five-week-old control chickens
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction was conducted as described above for channel 1. Equal amounts of total RNA from six control birds were combined to form a reference RNA pool.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
23 aliquots of the reference RNA were labelled with Alexa Fluor 647 (red) as decribed above for channel 1. A pool was made of all Alexa Fluor 647 -labeled cDNA to be split among the 23 slides in the reference hybridization experiment.
|
| |
|
| |
| Hybridization protocol |
After pre-hybridization of the microarray slides in blocking reagent (1% BSA, 5 x SSC, 0.2% SDS), each slide was hybridized with a sample labeled with Alexa Fluor 555 (green) and a reference RNA sample labeled with Alexa Fluor 647 (red) in a reference hybridization design. Slides were hybridized overnight in a 42 degree C water bath under a light-tight box. On the following day, slides were sequentially washed with 1 x SSC, 0.2% SDS, and 0.5% DTT at 55 degrees C for 10 min, then 0.1 x SSC, 0.2% SDS, and 0.5% DTT for 5 min at room temperature, and finally 0.1 x SSC and 0.5% DTT for 1 min at room temperature, with nitrogen gas bubbled through each of the washes to prevent interference of ozone. The slides were subsequently rinsed in dH2O and dried by centrifugation. Before scanning, slides were stored in individual 50 ml tubes covered in aluminum foil and filled with nitrogen gas.
|
| Scan protocol |
Slides were scanned using an Axon 4100A Microarray Scanner (Axon Instruments) with GenePix Pro 5.0 software (Axon Instruments) and at a PMT count ratio (635/532) of about 1.
|
| Description |
Chicken day six liver, Vehicle Control replicate 6
|
| Data processing |
The results of image analysis were automatically merged with Excel files containing the clone identification number/plate address and gene name/function (from the highest BLAST score). Data in the VALUE column is based on the loess-normalized ratio of the loess-normalized log2 Alexa 555 and Alexa 647 values. The values are based on non-background corrected median values.
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| |
|
| Submission date |
Jun 30, 2006 |
| Last update date |
Aug 08, 2006 |
| Contact name |
Larry Albert Cogburn |
| E-mail(s) |
cogburn@udel.edu
|
| Phone |
302-831-1335
|
| Organization name |
University of Delaware
|
| Department |
Animal and Food Sciences
|
| Lab |
Avian Functional Genomics
|
| Street address |
531 South College Ave.
|
| City |
Newark |
| State/province |
DE |
| ZIP/Postal code |
19717 |
| Country |
USA |
| |
|
| Platform ID |
GPL1731 |
| Series (1) |
| GSE5205 |
Analysis of Hepatic Gene Expression in Chickens with Hormonally-Induced Lean and Fat Phenotypes |
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