Total RNA was extracted using the mirVana miRNA Isolation Kit (Cat. AM1561, Invitrogen, Thermo Fisher Scientific Inc., USA) following the manufacturer’s protocol. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
The sample labeling, microarray hybridization and washing were performed based on the manufacturer’s standard protocols. Briefly, total RNA were dephosphorylated, denatured and then labeled with Cyanine-3-CTP.
Hybridization protocol
After purification the labeled RNAs were hybridized onto the microarray.
Scan protocol
After washing, the arrays were scanned with the Agilent Scanner G2505C (Agilent Technologies).
Data processing
Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. Next, GeneSpring software (version 14.8, Agilent Technologies) was employed to finish the basic analysis with the raw data. The raw data was normalized with the quantile algorithm.
