tissue: abdominal adipose genotype: LG cockerel age: 7 week of age
Biomaterial provider
SRA-INRA, Nouzilly, France
Treatment protocol
Divergently selected on body weight (HG genotype).
Growth protocol
Standard broiler chicken growth protocol
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a RNeasy Midi kit according to the manufacturer’s protocol (Qiagen; Valencia, CA). The RNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE). RNA integrity was examined using a RNA 6000 Nano Assay kit and the Model 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA).
Label
Alexa Flour 647
Label protocol
Twenty micrograms of total RNA were indirectly labeled using SuperScript Plus Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA). Briefly, the first strand cDNA synthesis was performed in a 30 microliter final volume containing 1x first-strand buffer, 5 micrograms of anchored oligo(dT)20, DTT, dNTP mix (including aminoallyl- and aminohexyl-modified nucleotides), 40 U of RNaseOUT and 800 U of SuperScript III reverse transcriptase with an incubation at 46 degrees Celsius for 3 hours. The original RNA was destroyed by NaOH hydrolysis and followed by neutralization with HCl. The cDNAs were purified using a low-elution volume spin cartridge (Invitrogen; Carlsbad, CA) and labeled with either Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester in the dark at room temperature for 2 hours. After purification of labeled cDNAs with a low-elution-volume spin cartridge, the efficiency of dye incorporation was determined using the Microarray Module on the NanoDrop ND-1000 spectrophotometer and the Base:Dye Ratio Calculator on the Invitrogen website.
tissue: abdominal adipose genotype: HG cockerel age: 7 week of age
Biomaterial provider
SRA-INRA, Nouzilly, France
Treatment protocol
Divergently selected on body weight (HG genotype).
Growth protocol
Standard broiler chicken growth protocol
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a RNeasy Midi kit according to the manufacturer’s protocol (Qiagen; Valencia, CA). The RNA concentration was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE). RNA integrity was examined using a RNA 6000 Nano Assay kit and the Model 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA).
Label
Alexa Flour 555
Label protocol
Twenty micrograms of total RNA were indirectly labeled using SuperScript Plus Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA). Briefly, the first strand cDNA synthesis was performed in a 30 microliter final volume containing 1x first-strand buffer, 5 micrograms of anchored oligo(dT)20, DTT, dNTP mix (including aminoallyl- and aminohexyl-modified nucleotides), 40 U of RNaseOUT and 800 U of SuperScript III reverse transcriptase with an incubation at 46 degrees Celsius for 3 hours. The original RNA was destroyed by NaOH hydrolysis and followed by neutralization with HCl. The cDNAs were purified using a low-elution volume spin cartridge (Invitrogen; Carlsbad, CA) and labeled with either Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester in the dark at room temperature for 2 hours. After purification of labeled cDNAs with a low-elution-volume spin cartridge, the efficiency of dye incorporation was determined using the Microarray Module on the NanoDrop ND-1000 spectrophotometer and the Base:Dye Ratio Calculator on the Invitrogen website.
Hybridization protocol
A cDNA spotted glass slide (Del-Mar 14K Chicken Integrated Systems microarray) was pre-treated for 45 min at 42 degrees Celsius in pre-hybridization solution containing 5x SSC, 0.1 % SDS and 1 % BSA and followed by dipping for 5 min in deionized water and dry spinning for 5 min at 1,000 x g. The labeled cDNA was dried down (Speed Vac) and reconstituted prior to hybridization in a 30 microliter volume of pre-warm DIG Easy Hyb solution (Roche Diagnostics; Indianapolis, IN). The two target samples (Alexa 555 and Alexa 647 labeled) were pooled and mixed with 2.5 microliter of 10 mg/ml Yeast tRNA, 2.5 microliter of 10 mg/ml salmon testes DNA (Sigma, Louis, MO) and 2.5 microliter of 1 microgram/microliter of pSport 6.1(Invitrogen) vector (PCR amplified product from “empty” pSport 6.1 vector sequence). The target mixture was denatured for 2 min at 94 degrees Celsius, cooled at room temperature, carefully loaded onto the middle of a pre-treated mircoarray slide held in a hybridization chamber (Corning #2551, Lowell, MA) and overlaid with a 22 x 65 mm LifterSlip (Erie Scientific, Portsmouth, NH). The sealed hybridization chamber was incubated overnight (approximately 16 hours) at 42 degrees Celsius in a 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 50 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 from ozone. The slides were subsequently rinsed in dH2O and dried by centrifugation. Slides were stored until scanning in individual 50 ml tubes covered in aluminum foil and filled with nitrogen gas.
Scan protocol
Each microarray slide was scanned using a GenePix 4000B microarray scanner (Molecular Devices; Sunnyvale, CA) set at 635 nm for Alexa647 and 532 nm for Alexa555 after adjustment of the PMT count ratio (635/532) to 1. Two high-resolution TIFF images (embedded in a single file) were generated for each slide.
Description
Chicken Abdominal fat, high (HG) versus low (LG) growth lines, 7 week of age, replicate 3
Data processing
Image analysis was performed on high-resolution TIFF images using GenePix Pro 4.1 (Molecular Devices, Sunnyvale, CA). The results of image analysis [GenePix 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). Fluorescence intensities were analyzed with the linear mixed model (MIXED) procedure in SAS (SAS Institute Inc., Cary, NC, USA).
Transcriptional profiling of abdominal adipose tissue in juvenile broiler chickens divergently selected for high or low body weight
Data table header descriptions
ID_REF
VALUE
Values supplied are the loess-normalized ratio (the base 2 power of (Alexa 647 loess adjusted median - Alexa 555 loess adjusted median)). The values are based on median intensity values (not background corrected) for Alexa 555 and Alexa 647.
