Total RNAs were extracted by the PicoPure RNA isolation kit (Arcturus/Molecular Devices).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low RNA Input Linear Amplification kit.(Cat#5184-3523, Agilent technologies, Santa Clara, CA, US), 5-(3-aminoallyl)-UTP (Cat#AM8436, Ambion, Austin, TX,US), Cy3 NHS ester (Cat#PA13105,GE healthcare Biosciences, Pittsburgh, PA,US) followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies,Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US),according to the manufacturer’s instructions. After 17 hours 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), Stabilization and Drying Solution (Cat#5185-5979, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions.
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，Scan resolution=5μm, PMT 100%，10%.
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
