Gene chip expression analysis
Gene chip expression analysis was performed by Kangcheng Biotechnology
Co., Ltd. (Shanghai, China) using the Agilent array platform. First, the
RNA quantity and quality were assessed using a NanoDrop ND-1000 UV
Spectrophotometer (Implen, German). RNA integrity of each sample was
identified by standard denaturing agarose gel electrophoresis. Second,
sample labeling and chip hybridization were performed according to the
Agilent One-Color Microarray-Based Gene Expression Analysis protocol
(Agilent, California, USA). The samples were labeled by using the
Agilent Quick Amp Labeling kit and hybridization experiments were
performed by using the Agilent SureHyb. Specifically, the total RNA of
each sample was linearly amplified and labeled with Cy3-UTP; the labeled
cRNAs were purified by using the RNeasy Mini Kit (Qiagen, Dusseldorf,
German), and the concentration and activity were detected with a
NanoDrop ND-1000 UV spectrophotometer. The purified Cy3-UTP-labeled
cRNAs were hybridized with Agilent mouse 4×44K gene expression profiling
chip v2 (Agilent, California, USA). The hybridization chip was washed,
fixed, and scanned by using Agilent DNA Microarray Scanner (part number
G2505C); Chip probe signal values were acquired by using Agilent Feature
Extraction software (v11.0.1.1) to obtain raw data. Finally, the
Quantile normalization of raw data and subsequent data processing were
performed by using GeneSpring GX v12.1 software (Agilent, California,
USA).