SK-N-MC cells were seeded at 5x104 cells/cm2 and grown for 24 h. BB or RV was supplied to cells (0.5 µg GAE/mL) dissolved in medium containing 0.5% (v/v) FBS for 24 h. 300uM of hydrogen peroxde was provided or not to corresponding samples for 3h. After incubation, cells were washed with PBS, tripsinized, collected to a tube and 0.05 volumes of FBS added, to inactivate trypsin. Dihexyloxacarbocyanine iodide (DiOC6(3)) and PI were added to cells to a final concentration of 20 nM and 1 µg/mL, respectively, and incubated for 30 min at 37 ºC. DiOC6(3) was used to evaluate the mitochondrial transmembrane potential (ΔΨm) and PI to determine cell viability, based on plasma membrane integrity. Cells were then sorted in a FACSAria High Speed Cell Sorter (Becton Dickinson), using a 100 µm nozzle with 206.8 kPa (30 psi) sheath pressure. A 488 nm laser was used for DiOC6(3) and PI excitation and detection was performed using a 530/30 nm and a 695/40 nm HQ band pass filter, respectively. Only cells that presented cellular membrane integrity and high mitochondrial transmembrane potential were collected in tubes containing PBS for further analysis.
Growth protocol
SK-N-MC cells (ECACC) were grown in EMEM supplemented with 10% FBS, 1% sodium-pyruvate, 1x Non-essential amino acids, 200mM glutamine, 1% pen-strep
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction of sorted cells was performed using AxyPrep Multisource Total RNA Miniprep (Axygen). Total RNA was treated with Turbo™ DNase I (Ambion), accordingly to the manufacturer’s instructions. RNA quantity was assessed using a Nano-Drop® ND-1000 spectrophotometer (NanoDrop Technologies) and RNA quality using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Assay (Agilent Technologies).
Label
biotin
Label protocol
Biological triplicates were processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip HuGene 1.0 ST Arrays in the Gene Expression Unit of Instituto Gulbenkian de Ciência (Oeiras, Portugal), and, according to the manufacturer’s Whole Transcript Sense Target Labeling Assay. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (GeneChip® WT cDNA Synthesis Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription reaction to generate cRNA (GeneChip® WT cDNA Amplification Kit; Affymetrix). Fifteen µg of this cRNA was used for a second cycle of first-strand cDNA synthesis (GeneChip® WT cDNA Synthesis Kit; Affymetrix). From the single stranded cDNA 5.5 µg was fragmented and end-labeled (GeneChip® WT Terminal Labeling Kit; Affymetrix). Size distribution of the fragmented and end-labeled cDNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
Hybridization protocol
Five µg of end-labeled, fragmented cDNA was used in a 100-µL hybridization cocktail containing added hybridization controls. Eighty µL of mixture was hybridized on arrays for 17 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0007; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
Scan protocol
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
Description
S4
Data processing
Scanned arrays were analyzed first with Affymetrix Expression Console software for quality control. For subsequent analyses Chipster 2.0 (http://chipster.csc.fi/) was used with custom cdf file HuGene10stv1_Hs_ENTREZG.cdf as available from Brainarray database version 14.1.0. Array raw data were normalized using Robust Multi-array average (RMA) and statistical differences evaluated by a two groups test (empirical Bayes), for p<0.05.
