Cell lines were grown in a Dulbecco´s Modified Eagle´s medium/Nutrient Mixture F-12 Ham (DMEM/F12) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Human oral epithelial tissue was obtained from healthy volunteers undergoing dental surgeries, following previously published procedures, after approval by the Research Ethics Committee of IPEN under License Number 087/CEP-IPEN/SP and with informed consent signature. Keratinocytes were grown on a fibroblast feeder-layer in a Dulbecco’s Modified Eagle Medium (DMEM; Gibco, New York, NY, USA) F-12 Nutrient Mixture (HAM, Gibco, New York, NY, USA) (2:1), with 10% Bovine Serum Product Fetal Clone III (Hyclone, Logan, Utah, USA), penicillin (100 U/ml), streptomycin (100 lg/ml), gentamicin (50 lg/ml), and amphotericin B (2.5 lg/ml) glutamine (4 mM), adenine (0.18 mM) (Sigma–Aldrich, St. Louis, MO, USA), insulin (5 lg/ml) (Sigma–Aldrich, St Louis, MO, USA), hydrocortisone (0.4 lg/ml) (Sigma–Aldrich, St Louis, MO, USA), cholera toxin (0.1 nM) (Sigma–Aldrich, St Louis, MO, USA), triiodotyronine (20 pM) (Sigma–Aldrich, St Louis, MO, USA) and epidermal growth factor (10 ng/ml) (R&D Systems, Minneapolis, MN, USA)
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from cell culture using mirVana miRNA Isolation Kit (Ambion Inc.) in compliance with the manufacturer’s protocol. RNA integrity and concentration were assessed using the RNA 6000 Nano Assay kit with Agilent 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoVue (GE Healthcare) Spectrophotometer.
Hybridization protocol
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on theGenePix Scanner using settings suggested by Agilent
Description
Gene expression
Data processing
The scanned images were analyzed with Feature Extraction Software v.9.5 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
High-throughput sequencing of small RNA transcriptomes and DNA microarrays reveal critical biological features targeted by microRNAs in cell models used for squamous cell cancer research
