Nanodrop technology nd 1000

Peripheral blood mononuclear cells (PBMCs) were either isolated from fresh blood by Ficoll-Hypaque gradient centrifugation or used after thawing. PBMCs (20×10⁶ cells) were cultured in RPMI medium containing 10% FBS and 2% gentamycin. Cells were stimulated with 1 ug/ml of phytohaemagglutinin (PHA; Sigma-Aldrich, St. Louis, Mo, USA) for 72 hours, washed in PBS (1×) and pelleted for RNA extraction. Total RNA (enriched for small RNA) was isolated according to manufacturer's instructions using the mirVana miRNA isolation Kit (Ambion, Huntingdon, UK). RNA concentration was calculated using NanoDrop technology ND-1000 (Thermo Scientific, Waltham, MA, USA). RNA integrity was then evaluated using RNA 6000 Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All chips were prepared according to the manufacturer's instructions at the Genomic platform of the CCiTUB (Centres Científics i Tecnològics University of Barcelona) located at the Barcelona Science Park (PCB). Total RNA degradation was evaluated by reviewing the electropherograms and the RNA integrity number (RIN) of each sample. Only samples with preserved 18S and 28S peaks and RIN values greater than 7 were selected for miRNA profile analysis.

DNA was extracted using the phenol/chloroform protocol [65 ] and purified through Amicon Ultra-0.5 mL Centrifugal Filters (EMD Millipore) to achieve a DNA concentration of 100 ng/µL. DNA concentration was checked via NanoDrop technology (ND-1000, Thermo Fisher Scientific) and Qubit Quantitation technology (High Accuracy & Sensitivity, Thermo Fisher). DNA methylation analysis was executed in two phases: the EZ DNA Methylation kit (Zymo Research) was used to perform sodium bisulfite conversion; the Illumina Infinium Methylation EPIC Chip (with over 850,000 methylation sites; Illumina Inc.) was used to quantify DNA methylation according to the standard Illumina protocol. The methylation array analysis was performed at the Connecting bio-research and Industry Center, Trieste—Italy.