|
| Status |
Public on Nov 12, 2007 |
| Title |
Chicken hypothalamus, lean line, 9 weeks of age, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Chick hypothalamus
|
| Organism |
Gallus gallus |
| Characteristics |
Chicken hypothalamus lean line cockerel, 9 weeks of age
|
| Biomaterial provider |
SRA-INRA, Nouzilly, France
|
| Treatment protocol |
Divergently selected for leanness.
|
| Growth protocol |
Male chickens used in this study were from lean line, which were derived from a population of broiler chickens divergently selected for high and low abdominal fat content (Leclercq et al., 1980). Tissues were excised immediately after death, snap frozen in liquid nitrogen, and stored at -80 degree Celsius until RNA isolation. The LL cockerels were raised in floor pens in a 4.4 x 3.9 m2 room. Pelleted feed (22% crude protein and 3100 kcal ME/kg) was provided throughout the experiment. After continuous light for the first two days, the birds were maintained on a 14L:10D light/dark cycle. Supplemental heat was provided by infrared gas and ambient temperature was progressively decreased from 32 degree Celsius at hatching until 22 degree Celsius was reached at 22 days. The birds were provided with water and feed ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from frozen tissue samples 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 ribosomal RNA ratio (28S/18S) was observed for integrity.
|
| Label |
Cy3
|
| Label protocol |
For indirect labeling, 25 micrograms of total RNA was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), and Cy3-monoreactive NHS esters (Amersham Biosciences, Piscataway, NJ) were coupled to the cDNA. Labeled cDNA was purified from unincorporated dye using CyScribe GFX Purification Kit (Amersham Biosciences).
|
| |
|
| Channel 2 |
| Source name |
Multi-tissues reference RNA pool
|
| Organism |
Gallus gallus |
| Characteristics |
Multi-tissue RNA pool was made from an equal amount of total RNA from chicken liver, hypothalamus, breast muscle and abdominal fat.
|
| Biomaterial provider |
Moyer's Hatchery (Quakertown, PA) for liver, breast muscle and abdominal fat and SRA-INRA (Nouzilly, France) for (hypothalamic samples.
|
| Treatment protocol |
Control broiler cockerels were used as a source of liver, breast muscle and abdominal fat. Lean line chickens were used as a source for hypothalamic samples. Tissues were excised immediately after death, snap frozen in liquid nitrogen, and stored at -80 degrees Celsius until RNA isolation.
|
| Growth protocol |
Control Broiler chicks (Ross x Cobb)were raised under conditions described above for channel 1. Lean line chickens were derived from a population of broiler chickens divergently selected for high and low abdominal fat content (Leclercq et al., 1980).The lean line cockerels were raised in floor pens in a 4.4 x 3.9 square meter room. Pelleted feed (22% crude protein and 3100 kcal ME/kg) was provided throughout the experiment. After continuous light for the first two days, the birds were maintained on a 14L:10D light/dark cycle. Supplemental heat was provided by infrared gas and ambient temperature was progressively decreased from 32 degrees Celsius at hatching until 22 degrees Celsius was reached at 22 days. The birds were provided with water and feed ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from frozen liver and hypothalamus samples was isolated using a RNeasy Midi kit (Qiagen, Valencia, CA) following the manufacturer's protocol with some modifications. Total RNA from breast muscle and abdominal fat was isolated using the guanidine thiocyanate lysis method and CsCl gradient purification. 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 ribosomal RNA ratio (28S/18S) was examined for integrity.
|
| Label |
Cy5
|
| Label protocol |
For each sample, a 25 microgram aliquot of reference RNA pool was labeled with Cy5 (red) as described above for channel 1. For each hybridization set, a final reference pool was made of all Cy5-labeled cDNA and equally aliquoted for all slides hybridized that day.
|
| |
|
| |
| Hybridization protocol |
The microarray was hybridized overnight at 42 degrees Celsius with Cy3-labeled cDNAs from the target sample and an aliquot of the Cy5-labeled reference RNA pool cDNAs using microarray hybridization buffer (Amersham Biosciences). On the following day, slides were sequentially washed with 1 x SSC, 0.2% SDS at 50 degrees Celsius for 10 min, then 0.1 x SSC and 0.2% SDS for 5 min at room temperature, and finally 0.1 x SSC for 1 min at room temperature. The slides were subsequently rinsed in dH2O and dried by centrifugation.
|
| Scan protocol |
The microarray slides were scanned using a 418 confocal laser scanner (Affymetrix) at 550 nM for Cy3 and 649 nM for Cy5 to generate two TIFF images for each slide. The photomultiplier tube (PMT) gain for the Cy3/Cy5 ratio was set to 1. For the high resolution scan for each dye, the PMT gain was set to achieve 1% saturation.
|
| Description |
Hypothalamus from lean line chickens, replicate 2
|
| Data processing |
For each microarray the high-resolution TIFF image files were analyzed using GenePix Pro V4.1 software (Axon Laboratories, Palo Alto, CA). The GenePix Pro report (gpr) files 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 Cy3 and Cy5 values. The values are based on non-background corrected median values.
|
| |
|
| Submission date |
Oct 26, 2007 |
| Last update date |
Aug 14, 2011 |
| 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) |
| GSE9500 |
Utility of the Del-Mar 14K Chicken Microarray for Transcriptional Profiling in Different Orders of the Class Aves |
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