Confluent cultures were transferred to fresh Dulbecco’s modified Eagle’s medium, which contained 50 ng/mL insulin, 0.25 μM dexamethasone, 5 mM octanoate, 10 mM acetic acid, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured for up to 6 days and the medium was changed every 2 days. BIP cells were treated with ATRA (1 μM), 9cRA (1 μM), or they received no treatment (control).
Growth protocol
BIP cells were cultured according to previously reported methods (Aso et al. 1995, Mizoguchi et al. 2014).
Extracted molecule
total RNA
Extraction protocol
BIP cells were harvested at 0 (D0) and 6 days (D6) after adipogenic stimulation. Total RNA samples were extracted from BIP cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then treated with DNase I (Takara Bio Inc., Shiga, Japan).
Label
Cy3
Label protocol
The total RNA was amplified and labeled with Cyanine 3 (Cy3) using an Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions. In brief, 100 ng of total RNA was reverse transcribed to double-stranded cDNA using a poly dT-T7 promoter primer. The primer, template RNA, and quality-control transcripts with known concentrations and quality were denatured at 65degC for 10 min and then incubated for 2 h at 40degC with 5× First-Strand Buffer, 0.1 M dithiothreitol, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was inactivated at 70degC for 15 min and the cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA. The cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and then incubated at 40degC for 2 h. The labeled cRNAs were purified using RNeasy mini spin columns (QIAGEN GmbH, Hilden, Germany) and eluted in 30 μL of nuclease-free water.
Hybridization protocol
After amplification and labeling, cyanine incorporation and the cRNA quantity were determined using an Agilent Bioanalyzer and a Nanodrop ND-1000 spectrophotometer. For each hybridization, 1.65 μg of Cy3-labeled cRNA was fragmented and hybridized at 65degC for 17 h to an Agilent Bos taurus (Bovine) Oligo Microarray v2 (Design ID: 023647).
Scan protocol
The microarrays were scanned using an Agilent DNA microarray scanner.
Data processing
The intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtraction. We only used the features that were flagged as having no errors (flags present), whereas we excluded features that were not positive, not significant, not uniform, not above the background, saturated, or population outliers (marginal and absent flags).
Microarray analysis indicates that vitamin A alters expression profiles of bovine intramuscular preadipocytes during adipogenesis
Data table header descriptions
ID_REF
VALUE
Normalization was performed using Agilent GeneSpring GX version 11.0.2. (per chip: normalization to 75th percentile shift; per gene: normalization to median of all samples).
