gender: female, age: 60 weeks, strain: pedigree line Hy-Line White Leghorn layer, tissue: crude bone marrow sample
Biomaterial provider
Hy-Line International
Treatment protocol
none
Growth protocol
Birds were obtained on day of hatch and fed a starter diet to 6 wks of age, a grower diet from 6 to 8 wks, a developer diet from 8 to 15 wks, a pre-lay diet from 15 to 18 wks, and a breeder diet from 18 wks until termination of the experiment. Layers were provided feed and water ad libitum, however daily feed restriction beginning at 6 wks, based on average body weights collected at monthly intervals, was necessary to prevent obesity in the broilers. All animal management procedures were approved by the Purdue University Animal Care and Use Committee.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from entire bone marrow samples with Trizol Reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s recommended protocol.
Label
Alexa Fluor 647
Label protocol
Forty µg of bone marrow RNA was purified and concentrated using the RNase-Free DNase Set along with the RNeasy MinElute Cleanup Kit (QIAGEN Incorporated, Valencia, CA). Twenty µg of total RNA was reverse transcribed into first-strand cDNA with oligo(dT)20 and random hexamer primers, then fluorescently labeled with either Alexa Fluor 555 or 647 dye using the SuperScript Plus Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) with the exception that the PureLink PCR Purification Kit (Invitrogen Corporation, Carlsbad, CA) was used for the purification steps.
Hybridization protocol
The Fred Hutchinson Cancer Research Center (FHCRC) Chicken 13K array (Fred Hutchinson Cancer Research Center, Seattle, WA) was used to compare gene expression profiles from the layer and broiler lines. Each array was pre-hybridized, hybridized, and washed per manufacturer’s recommended protocols (Burnside et al., BMC Genomics 6: 13, 2005), with the exception that wash 2 and 3 times were cut in half. One layer and one broiler sample labeled with different dyes were hybridized to each array (6 arrays per time point) in 50 µl of hybridization buffer solution using Lifterslip (Erie Scientific Company, Portsmouth, NH) cover slips, and incubated in a 63°C water bath within individual hybridization chambers wrapped in foil to block out light to prevent dye bleaching from light exposure.
Scan protocol
Slides were scanned with array WoRxe Biochip Reader (Applied Precision, LLC, Issaquah, WA) and images analyzed with ImaGene (BioDiscovery Incorporated, El Segundo, CA) software.
Description
Following euthanasia at 15 weeks of age, the right tibia was excised and cracked with a hammer. Bone marrow was collected using a metal spatula from the entire length of the bone, frozen in liquid nitrogen and maintained in a -80 degree Celsius freezer pending RNA extraction.
Data processing
Three layer and three broiler samples were labeled with each dye to control for dye bias. Features with an intensity level greater than two times its local background for at least one of the 12 channels (6 dual channel slides) in the dataset were retained. Using the statistical software package R, these data were log transformed and normalized using lowess normalization and median centering across all hybridizations (Yang et al., Nucleic Acids Res. 30(4): e15, 2002).
Differential gene expression in the bone marrow of layer and broiler chickens
Data table header descriptions
ID_REF
VALUE
Features with an intensity level greater than two times its local background for at least one of the 12 channels (6 dual channel slides) in the dataset were retained. Using the statistical software package R, these data were log transformed and normalized using lowess normalization and median centering across all hybridizations.
