PBMC were isolated by density separation over a Ficoll-PaqueTM (GE healthcare, Uppsala, Sweden) gradient (460g for 30 min) and were washed three times with PBS pH 7.4/1 mM EDTA (Sigma, Milan, Italy). Cells were counted and their viability was evaluated by trypan blue exclusion (>90% PBMC were viable).Total RNA was extracted from a minimum of 106 PBMCs using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The concentration of total RNA was measured using NANODROP® spectrophotometer ND-1000 (Thermo Scientific, Waltham, MA, US) and RNA integrity was measured using the Agilent 2100 BIOANALYZER® (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) along with the Agilent® RNA 6000 Nano Kit.
Label
Cy3
Label protocol
Labeled cRNA was generated using the Low Input Quick Amp Labeling (LIQA) kit (Agilent Technologies, Santa Clara, CA).
Hybridization protocol
Labeled cRNA was then hybridized for 17 hours at 65°C on Whole Human Genome Microarray 4x44 K slides, (Agilent Technologies, Santa Clara, CA). After hybridization the slides were washed according to manufacturer’s protocols.
Scan protocol
The slides were scanned using the High-Resolution Microarray C Scanner (Agilent Technologies).
Data processing
Data extraction was performed using Agilent Feature Extraction software. A p-value cut-off of 0.05 and a fold-change cut-off of 2 were applied to identify differentially expressed genes able to discriminate between the study groups.
