Total RNA was extracted and purified using mirVana™ miRNA Isolation Kit (Cat#AM1560, Ambion, Austin, TX, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100(Agilent technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)in hybridization Oven(Cat#G2545A, Agilent technologies, Santa Clara, CA, US)at 55℃，20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner(Cat#G2565BA，Agilent technologies, Santa Clara, CA, US)and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US)with default settings.
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0(Agilent technologies, Santa Clara, CA, US).
