cell type: Ntera2/D1 agent: Retinoic Acid time: 6 Days
Biomaterial provider
Stratagene, La Jolla, CA
Treatment protocol
The cells were seeded at a density of 2 × 106 cells per T75 flask and treated with 10µM all-trans retinoic acid (RA, Sigma, Oakville, Ontario, Canada) in fresh medium every two days during a 28-day differentiation timecourse. Cells were harvested by tripsinization and centrifugation 0, 2, 4, 6, 8, 12, 14, 21, and 28 days following addition of RA. Time-course experiments were repeated several times to ensure multiple biological replicates for microarray analysis.
Growth protocol
Human embryonal teratocarcinoma Ntera2/D1 (NT2) cells (Stratagene, La Jolla, CA) were cultured in high glucose Dulbecco’s modified Eagle’s medium (HG/DMEM, Invitrogen, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS, Wisent, Saint-Jean, Quebec, Canada).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the cells using TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. High molecular-weight RNAs were removed by precipitation with 12.5% PEG-8000 and 1.25 M NaCl.
Label
Cy3
Label protocol
RNA labelling and hybridization to the microarrays was carried out using a method adapted from Thomson et al. 2004 (Thomson JM, Parker J, Perou CM, Hammond SM. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat Methods 1: 47-53). Briefly, 10 or 25 µg total RNA was PEG-precipitated (as above), resuspended in 6 µl water, and mixed into a labelling reaction (final volume 10 µl) consisting of 500 ng 5’-phophate-cytidyl-uridyl-Cy3-3’ (Dharmacon, Chicago, IL), 10% DMSO, 0.1 mM ATP, 50 mM HEPES (pH 7.8), 3.5 mM DTT, 20 mM MgCl2, 0.1 µg BSA, and 20 U T4 RNA ligase (NEB, Ipswitch, MA). Reactions were incubated on ice in the dark for 2 hours before precipitation with 0.27 M sodium acetate, 20 µg glycogen, and 2.7 volumes ethanol in a 200 µl final volume. Following 10 min incubation on ice and 10 min centrifugation, the pellet was washed in 70% ethanol, centrifuged 5 min, briefly dried, and resuspended in 6 µl water.
Hybridization protocol
Labelled RNA samples were hybridized to microarrays in a 35 µl final volume composed of 400 mM Na2HPO4 (pH 7.0), 5% SDS, 0.8% BSA, and 12% formamide. Hybridisation mixtures were denatured at 95°C for 4 minutes and immediately pipetted onto glass slides covered with 22×25 mm LifterSlips (Erie Scientific Company, Portsmouth, NH). Hybridizations were performed in custom-made sealed chambers submerged in a 37°C water bath overnight (18 hours). Slide washes were 3 min long: once in 2X SSC/0.025% SDS, three times in 0.8X SSC, and twice in cold 0.4X SSC. Immediately following the last wash, slides were dried by centrifugation.
Scan protocol
Slides were scanned with a ScanArray 5000XL (Packard BioScience, Meriden, CT) confocal scanner at 10 µm resolution.
Description
no additional information
Data processing
Microarray scans were analysed with QuantArray software version 3.0 (Packard BioScience, Meriden, CT) using an adaptive quantitation method. Spots of poor quality on the arrays were flagged for removal from the data processing. Cy3 mean pixel intensities were background subtracted, log transformed, and replicate spots on the arrays were averaged. Microarray data were normalised between arrays using a standard median centering procedure. miRNAs were considered expressed if their normalised log2 hybridisation signal was ≥ 2. Differentially-expressed miRNAs were identified using a SAM (significance analysis of microarrays) threshold of 5% and a fold-change of 2.
