Total circulating RNA was extracted from 200 µl of plasma using the Mirneasy serum/plasma advanced kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To improve RNA extraction, MS2 carrier (Merck Life Science S.r.l., Milan, italy) was added to each sample before lysis step. RNA elution was performed in 30 µl of nuclease-free water.
Label
Cy3
Label protocol
For miRNA profiling, 4 µl of totRNA of each sample were used and individually processed with the miRNA Complete Labeling and Hybridization kit (Agilent Technologies S.p.A., Cernusco sul Naviglio, Milan, Italy).
Hybridization protocol
Hybridization was carried out on 8x60K oligonucleotides arrays miRbase V21 (Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Milan, Italy).
Scan protocol
After hybridization, washing and scanning, by Agilent G2505C scanner.
Data processing
Images were analyzed using Feature Extraction software v10.7. Raw data were processed by means of the limma R library, applying background correction (normexp) and quantile normalization, and averaging miRNA levels when they are represented with the same probe on the array.
