RNeasy Mini Kit (Qiagen, Hilden, Germany) were used to extract total cellular RNA. Before microarray hybridization, each RNA sample was quantified using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and qualified using Bioanalyzer RNA Nano 6000 chip (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process.
Hybridization protocol
0.6 μg of Cy3-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent SurePrint G3 Human GE 8×60K Microarray (Agilent Technologies, USA) at 65°C for 17 h.
Scan protocol
After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3.
Description
0.4 mM IBMX
Data processing
Scanned images are analyzed by Feature extraction10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature.
